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. 2014 Apr 13;16(3):265–278.e13. doi: 10.1016/j.neo.2014.03.001

CTHRC1 Acts as a Prognostic Factor and Promotes Invasiveness of Gastrointestinal Stromal Tumors by Activating Wnt/PCP-Rho Signaling1

Ming-Ze Ma ⁎,2, Chun Zhuang †,2, Xiao-Mei Yang ⁎, Zi-Zhen Zhang †, Hong Ma ⁎, Wen-Ming Zhang ‡, Haiyan You ⁎, Wenxin Qin ⁎, Jianren Gu ⁎, Shengli Yang ⁎, Hui Cao †,⁎, Zhi-Gang Zhang ⁎,⁎
PMCID: PMC4094796  PMID: 24726140

Abstract

Gastrointestinal stromal tumors (GISTs) are the major gastrointestinal mesenchymal tumors with a variable malignancy ranging from a curable disorder to highly malignant sarcomas. Metastasis and recurrence are the main causes of death in GIST patients. To further explore the mechanism of metastasis and to more accurately estimate the recurrence risk of GISTs after surgery, the clinical significance and functional role of collagen triple helix repeat containing-1 (CTHRC1) in GIST were investigated. We found that CTHRC1 expression was gradually elevated as the risk grade of NIH classification increased, and was closely correlated with disease-free survival and overall survival in 412 GIST patients. In vitro experiments showed that recombinant CTHRC1 protein promoted the migration and invasion capacities of primary GIST cells. A luciferase reporter assay and pull down assay demonstrated that recombinant CTHRC1 protein activated noncanonical Wnt/PCP-Rho signaling but inhibited canonical Wnt signaling. The pro-motility effect of CTHRC1 on GIST cells was reversed by using a Wnt5a neutralizing antibody and inhibitors of Rac1 or ROCK. Taken together, these data indicate that CTHRC1 may serve as a new predictor of recurrence risk and prognosis in post-operative GIST patients and may play an important role in facilitating GIST progression. Furthermore, CTHRC1 promotes GIST cell migration and invasion by activating Wnt/PCP-Rho signaling, suggesting that the CTHRC1-Wnt/PCP-Rho axis may be a new therapeutic target for interventions against GIST invasion and metastasis.

Abbreviations: CTHRC1, collagen triple helix repeat containing 1; DFS, disease-free survival; ECM, extracellular matrix; GIST, gastrointestinal stromal tumors; OS, overall survival; qRT-PCR, quantitative real-time polymerase chain reaction

Introduction

Gastrointestinal stromal tumors (GISTs) are mesenchymal neoplasms that usually arise in the stomach or small intestine and typically cause bleeding, anaemia and pain [1]. It is believed that GISTs originate from interstitial cells of Cajal [2] and may also derive from gastrointestinal smooth muscles or gut stem cells [3]. The pathological features of GISTs range from benign neoplasms to fatal sarcomas [1,4]. Most gastrointestinal stromal tumors stain positively for KIT [5,6], Ki67 [7] and anoctamin 1; exon mutations [6] in KIT or PDGFRA genes in approximately 80% or 10% of GISTs, respectively, have been demonstrated [1]. More than 60% of GIST patients can be cured by surgical resection [8,9]. The use of imatinib mesylate (Gleevec; Novartis) adjuvant treatment [8] is recommended in advanced GIST patients with postoperative recurrence risk, and the survival rate can be improved; however, secondary imatinib resistance is common [10].

Micrometastases and overt metastases are the main causes of death in malignant tumors, and this is the case in GIST patients as well [11,12]. Approximately 40% GISTs patients had metastatic lesions when definitively diagnosed, and more than 10% patients exhibited overt metastases [1]. Therefore, developing new predictors that can be used to estimate the risk of metastasis and postoperative recurrence is urgent.

Extracellular matrix (ECM) proteins play important roles in regulating tumor invasion and metastasis [13-15]. Given the secretary property, ECM proteins are also ideal candidates for tumor serum biomarkers and therapeutic targets.

Collagen triple helix repeat containing-1(CTHRC1) is a 28-kD extracellular matrix glycoprotein containing an NH2-terminal signaling peptide for extracellular secretion, a short collagen triple helix repeat of 36 amino acids, and a COOH-terminal globular domain [16].CTHRC1 was initially found in a screen for differentially expressed genes in balloon-injured versus normal rat arteries [16]. It has been reported that the CTHRC1 protein positively regulates the Wnt-PCP pathway by stabilizing formation of the Wnt ligand and Frizzled receptor complex [17] in developmental morphogenesis [17]. CTHRC1 has recently been shown to be highly expressed in human pancreatic cancer tissues [18], hepatocellular carcinoma [13], gastric cancer [19], and colorectal cancer [20], and it promotes invasion and metastasis in these cancers. Several studies revealed that CTHRC1 regulates cancer cell motility and invasiveness through activating the Wnt-PCP pathway [18]. However, the clinical significance and functional role of CTHRC1 in GIST remain unclear. In this study, we first examined the expression of CTHRC1 and its correlation with the clinicopathological parameters of GIST. Then, we further analyzed the relationship between CTHRC1 expression and the survival of GISTs patients and identified CTHRC1 as a novel prognostic factor of GIST. Finally, we demonstrated that CTHRC1 promoted migration and invasion of primary GIST cells through activated Wnt/PCP-Rho signaling.

Materials and Methods

Ethics Statement

We obtained approval from the Regional Ethical Committees, Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China for the use of clinical GIST patients' tissues. All the patients joined this study have signed informed consent. Ethical approval number, 2012031.

Patients

The inclusion criteria for our study were as follows: 1) a distinct pathologic diagnosis of GIST (CD117 positive in immunohistochemistry staining) ; 2) primary GIST cases without history of other solid tumors; 3) accepted radical surgery treatment without tumor residual; 4) without any chemotherapy, radiotherapy or other anti-cancer therapies before surgery; 5) availability of complete clinicopathologic and follow-up data; 6) obtained informed consent of patients and approval of the ethics committee of Renji Hospital for the use of samples. A total of 412 GIST cases, pathologic diagnosed and treated range from September 2004 to September 2013, were retrospectively identified from the hospitalization archives of Department of General Surgery, Renji Hospital, Shanghai, China. The paraffin-embedded tissue samples of these patients were used for tissue microarray construction and immunohistochemical staining. The clinicopathologic parameters include patients' age, gender, pathogenic site, histological type, tumor size (cm), number of mitoses/50 high-power fields (HPF), tumor rupture, mutation type and imatinib adjuvant treatment regimens. The risk of GIST behavior was classified into very low, low, intermediate, and high-risk categories according to the modified National Institute of Health (NIH) consensus [21,22]. In our study, the criterion of imatinib adjuvant therapy is at least twelve months uninterrupted drugs at a dose of 400mg/day. All the patients involved in our research accepted physical examination once a month during the first year after surgery and every six months thereafter. High risk GIST patients were accepted computed tomography (CT) or magnetic resonance imaging (MRI) of abdomen and pelvis at three-months intervals during the first three years after surgery, and subsequently at six-months intervals until five years after surgery. Complete follow-up data for GIST patients in cohort were available. Patients were followed until September 2013. Overall survival (OS) was defined as the time from surgery to death or the last follow-up examination. Disease free survival (DFS) was defined from the date of surgery until the detection of tumor recurrence or last observation.

Tissue Microarray Construction

Tissue microarrays were constructed by Suzhou Xinxin Biotechnology ( Xinxin Biotechnology Co, Suzhou, China). Tissue paraffin blocks of GIST samples were stained with hematoxylin-eosin to confirm the diagnoses and marked at fixed points with most typical histological characteristics under a microscope. Two 1.6 mm cores per donor block were transferred into a recipient block tissue microarrayer, and each dot array contained fewer than one hundred and sixty dots. Three-micron-thick sections were cut from the recipient block and transferred onto glass slides using an adhesive tape transfer system for ultraviolet cross linkage.

Immunohistochemistry Stain

The tissue microarray glass slides were baked at 55°C for one hour, and then de-paraffinized gradually through xylene, 50% xylene, gradient concentrations of ethanol until immersed in tap water. Tissue sections were blocked for peroxidase activity with 0.3% Hydrogen peroxide at 37°C for 30mins. Antigen retrieval was carried out via boiling in 10mmol/L citrate buffer (pH6.0) for fifteen mins. Then the tissues were incubated with CTHRC1 antibody (mouse monoclonal antibody, 1:100 dilution, Huaan Biotechnology, Hangzhou, China) overnight at 4°C. Next day, the tissues were washed with phosphate buffer solution (PBS) for three times and incubated with HRP-labelled anti-mouse secondary antibody (1:200dilution, Dako, Carpinteria , CA, USA) for one hour at room temperature. Immunostaining was carried out using diaminobenzidine substrate chromogen (Dako, Carpinteria, CA, USA) method and chromogenic reaction was controlled under microscope. After immunostaining, tissues were immersed into hematoxylin for nuclear staining. The TMA slides were then dehydrated through gradient concentrations of ethanol, cleared with xylene, and coverslipped with neutral balsam (Shenggong , Shanghai, China). The staining results were judged by two pathologists according to criterion as follows: 0: weak, no staining was observed; 1 +, 25% to 50% of the tumor cells were weak or moderate staining; 2 +, strong, more than 50% tumor cells were moderate or strong staining. 1 + and 2 + scores were identified as positive staining, while 0 score means negative staining. Negative controls for primary and secondary antibodies were shown in Figure W3. Total RNA Extraction and Quantitative Real-time PCR Total RNA was extracted from 29 fresh GIST tissues using Trizol reagent (Takara, Dalian, China) followed the manufacturer instructions. The reverse-transcription reactions were carried out with random primers and M-MLV Reverse Transcriptase (Takara, Dalian, China). The 29 cases of cDNA were used for templates of quantitative real-time PCR reaction in SYBR-Green method. All the qPCR reactions were performed on a StepOneTM real-time PCR System (Applied Biosystems, Foster City, CA,USA). Beta-actin was used as an internal control. The 2-△Ct method was used to quantify the relative CTHRC1 expression levels. The forward and reverse CTHRC1 primer sequences were: 5′-TGGTATTTCACATTCAATGGAGCTG-3′ and 5′-TGGGTA- -ATCTGAACAAGTGCCAAC-3′, respectively.

Western Blotting

Fresh GIST tissues were lysed in tissue protein extraction reagent (Invitrogen). Primary GIST cells were lysed in Western and IP lysis buffer (P0013, Beyotime, Jiangsu, China) supplemented with 1mM PMSF (Adamas beta, Shanghai, China). The lysis buffer includes, 20mM Tris (pH7.5), 150mM NaCl, 1%Triton X-100, sodium pyrophosphate, β-glycerophosphate, EDTA, Na3VO4, leupeptin. Proteins were separated by 10% SDS-PAGE under reducing condition, followed by blocking in phosphate-buffered saline/Tween-20 containing 1%BSA (Bovine Serum Albumin). The NC (Nitrocellulose filter membrane) or PVDF (Polyvinylidenefluoride) membrane was incubated with antibodies for CTHRC1(1:1000, mouse, Huaan, Hangzhou, China) , JNK (1:1000, Rabbit source, Cell Signaling Technology), RhoA (1:1000,Rabbit,CST), Rac1(1:1000,Mouse, Millipore) ,Cdc42 (1:1000,Mouse) and species-specific secondary antibodies. Bound the IRDye 680 anti-mouse (LI-COR, 1:20000) and IRDye 800 anti-rabbit (LI-COR, 1:10000) secondary antibodies were revealed by Odyssey imaging system (LI-COR). Wnt5a neutralizing antibody (R&D), Wnt3a neutralizing antibody (R&D), NSC23766 (Rac1 inhibitor, Merck Millipore, effective dose: 50 μM), Y-27632 (ROCK inhibitor, effective dose: 100 μM).

CTHRC1 Recombinant Protein Expression, Purification and Verification

CTHRC1 ORF were cloned into the episomal expression vector V152 (Figure W1B) with pCEP-Pu-Strep II-tag (C-terminal) in-frame and the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter. CTHRC1 was recombinant expressed in EBNA-293 cells after transfecting reconstructed plasmid by using X-tremeGENE 9 DNA Transfecting Reagent (Roche, Mannheim, Germany). Forty eight hours after transfection, the EBNA-293 cells were screening with puromycin (Sigma-Aldrich, St. Louis, MO) at a dose of 2μg/ml in DMEM supplemented with 10% FBS for seven days, then the culture media were collected and applied to the Strep Tactin sepharose column(IBA, Gottingen, Germany). After this, the column was washed with binding buffer and eluted by elution buffer containing 2.5 mM desthiobiotin. The collected fractions were further quantified by Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and BCA Protein Assay Kit (Pierce. Biotechnology Inc, Rockford, IL) and identified by western blotting assay.

In Vitro Migration and Invasion Assays

For the transwell migration assay, 4×104 primary GIST cells were placed on the top chamber of each insert with the noncoated membrane (Millicell). Cells were trypsinized and resuspended in DMEM and 700-900μL of medium supplemented with 10% fetal bovine serum added rCTHRC1 protein followed gradient doses of 0 nM, 1 nM, 20 nM, 50 nM respectively were injected into the lower chamber. After 24 hours for GIST cells in the migration assays, any cells remaining in the top chambers or on the upper membrane of the inserts were carefully removed. After fixation and staining in a dye solution containing 0.1% crystal violet and 20% methanol, cells adhering to the lower membrane of the inserts were counted and imaged through an IX71 inverted microscope (Olympus Corp. Tokyo, Japan). We carried out invasion assay by adding 100μl matrigel (BD Bioscience, Franklin Lakes, NJ) into top chamber of transwell and placed 8×104 primary GIST cells onto the matrigel. 48 hours later, the transwell for invasion was ceased and staining.

Cell Viability Assay

Cell viability was detected using a standard Cell Counting Kit-8 assay. Primary GIST cells were seeded into 96-well plates (100μl per well) at a density of 3×104 cells per ml. Cells in four divided groups were added rCTHRC1 protein followed gradient doses of 0 nM, 1 nM, 20 nM, 50 nM respectively. We added 10μl of reagent from Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) to each well for detection at day 1, 2, 3, 4, 5. After two hours of incubation at 37°C, the optical density was measured using microplate reader at a wavelength of 450nm.

Cell Isolation and Primary Cell Culture

Fresh surgical GIST tissues were gently minced with scissors, washed twice with phosphate-buffered saline (PBS), and then filtered through the steel mesh with 200 μm pore diameter. After washed in cold PBS, cell pellets were resuspended in RPMI-1640 medium supplemented with 20% fetal calf serum (FCS; Gibco, France) and seeded onto culture dishes. The primary GIST cells were cultured in incubator with 5%CO2 and 37 degrees centigrade. The culture medium for primary GIST cells were changed twice every three days . The successfully isolated primary GIST cells were shown in Figure 3A.

Figure 3.

Figure 3

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(A) Immunofluorescent staining showed the morphology of primary GIST cells isolated from the GIST tissues of three patients. The green fluorescence represents phalloidin for F-actin staining, whereas the blue fluorescence represents DAPI for nuclear staining. (B) Cell viability of primary GIST cells treated with rCTHRC1 protein at doses of 0 nM, 1 nM, 20 nM, 50 nM were measured using CCK-8 assay for six days. (C) Representative images (left) of GIST cells that migrated to the bottom of transwell filter (8μm, pore diameter) and statistical analysis (right) of the cell migration stimulated with rCTHRC1 protein or vehicle. (D) Representative images (left) of GIST cells that invaded through Matrigel to the bottom of transwell filter (8μm, pore diameter) and statistical analysis (right) of the cell invasiveness stimulated with rCTHRC1 protein or vehicle. The results shown are mean±SD of migration, and invading cells were photographed at 200 × magnification per field. (*, P < 0.05; **, P < 0.01).

Pull Down Assay

Pull down assays were conducted as reported [23] . Primary GSIT cells cultured in 100 mm dishes were serum-starved for 24 hours and treated with rCTHRC1 at a dose of 20 nM or desthiobiotin buffer for 2 hours. The primary antibodies used included the following: mouse primary antibody against Rac1 (Millipore, 1:1000) and rabbit primary antibody against RhoA, Cdc42 (Cell Signaling Technology, 1:1000).

Luciferase Reporter Assay

Primary GIST cells were seeded in 96-well plates and transfected with mixture of 100 ng TCF/catenin reporter plasmid (Wnt/β-catenin signaling), or 100 ng ATF2 reporter plasmid (Wnt/PCP signaling), and 10 ng Renilla following the recommended protocol for the Lipofectamine 2000 transfection system. One group of GIST cells were treated with rCTHRC1 protein at a concentration of 20 nM. After 48 hours of incubation, firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay system (Promega, Madison, WI) from the cell lysates.

Statistical Analysis

Statistical analyses were conducted using SPSS 16.0 software (Chicago, IL, USA). We performed chi-squared tests in cross tables to assess the relationships between expression levels of CTHRC1 and clinicopathological factors. Overall survival (OS) and Disease-free survival (DFS) were calculated using Kaplan-Meier method. The survival distributions were compared through log-rank test. All statistical tests were two-sided. One-way analysis of variance (ANOVA, Post-hoc testing) was used to compare groups (Table W1). P value less than 0.05 was considered statistically significant.

Results

CTHRC1 Expression Is Gradually Elevated in Accordance with GIST Risk Grades

To investigate the CTHRC1 expression level in GIST tumor tissues with different risk grades, we first evaluated the mRNA expression level of CTHRC1 in fresh GIST tissue samples by quantitative PCR (qPCR). The results showed that CTHRC1 mRNA expression levels in GIST tumor tissues of the intermediate- and high-risk groups were higher than those of the low-risk group (Figure 1A). We further compared the protein expression level of CTHRC1 in GIST tissues with different risk grades. The three low-risk, two intermediate-risk and five high-risk samples were analyzed by western blotting. The results showed that the CTHRC1 protein expression level in the high-risk group was significantly higher than that of the intermediate- and low-risk groups (Figure 1B).

Figure 1.

Figure 1

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CTHRC1 expression in GIST tissues. (A) Relative mRNA expression of CTHRC1 in low-risk group was significantly lower than those in the intermediate- and high-risk groups. (B) Western blotting analysis showed that CTHRC1 expression in high-risk GIST patients was significantly higher than those of low-risk or intermediate-risk groups. Tubulin was included as a loading control. (C) Representative image of immunohistochemical staining of CTHRC1 in low-risk, intermediate-risk and high-risk GIST tissues. Original magnification: a, b, c, 100 ×; d, e, f, 200 ×. Scale bars, 5 μm (*, P < 0.05).

CTHRC1 Protein Expression Level Is Closely Correlated with Risk Grade of NIH Classification, and Prognosis of GIST

The clinicopathological significance of CTHRC1 was further examined using a tissue microarray which contained 412 GIST tissue samples. The immunohistochemistry staining results showed that 145 (35.2%) cases showed CTHRC1 low expression, 267 (64.8%) cases showed CTHRC1 high expression (Figure 1C). The correlations between CTHRC1 expression and the clinicopathological parameters are shown in Table 1. We found that the expression level of CTHRC1 was higher in the patients with high NIH grade, large tumor size (> 10 cm) or increased mitotic figures than those with low NIH grade, small tumor size (< 10 cm), fewer mitotic figures with statistical significance (P < 0.05). Interestingly, we found that there was a significant difference between male (70.85%) and female (57.67%) GIST patients in frequency of high CTHRC1 levels. The statistical analysis suggested that CTHRC1 expression was not correlated with age, histological type, or tumor rupture. We further investigated the correlation between CTHRC1 expression and overall survival (OS) or disease-free survival (DFS) of GIST patients. The OS in the CTHRC1 negative (low) group (five-year OS rates, 100%, 145/145) was remarkably superior than that in the CTHRC1 positive (high) expression group (five-year OS rates, 90.6%, 242/267) (Figure 2A). The DFS in the CTHRC1 negative (low) group (five-year DFS rates, 95.2%, 138/145) was significantly higher than that in the CTHRC1 positive (high) expression group (five-year DFS rates, 76.8%, 205/267) (Figure 2B). In summary, CTHRC1 expression in GIST tumor tissues was closely correlated with OS and DFS of GIST patients.

Table 1.

Relationship between CTHRC1 expression and clinicopathologic features of GIST patients(*, P < 0.05; **, P < 0.01).

Variable CTHRC1(n = 412)
Low High P
Age$ ≤ 59 years 31 49 0.458
> 59 years 114 218
Gender Male 65 158 0.005
Female 80 109
Tumor site Stomach 100 128 < 0.001
Small bowel 28 93
Colon 12 9
Other sites 5 37
Tumor size(cm) ≤ 2 30 7 < 0.001
> 2&≤5 102 61
> 5&≤10 9 127
> 10 4 72
Mitoses per 50 HPFs ≤ 5 138 170 < 0.001
> 5&≤10 2 52
> 10 5 45
Modified NIH criteria Very low risk 30 2 < 0.001
Low risk 101 51
Intermediate risk 4 58
High risk 10 156
NIH invasion 1 30 2 < 0.001
2 101 51
3 4 58
4 5 101
5 5 55
Tumor bleeding No 131 220 0.030
Yes 14 47
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Abbreviations: HPF, high power field of the microscope; NIH, National Institutes of Health.

The P value in bold emphasize statistical significance (P<0.001).

$

Median age of total 412 patients was 59 years.

Figure 2.

Figure 2

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(A) Kaplan-Meier analysis of overall survival related to the expression of CTHRC1 in 412 GIST patients. (B) Kaplan-Meier analysis of disease-free survival related to the expression of CTHRC1 in 412 GIST patients. (C) Among CTHRC1-negative intermediate- and high-risk GIST patients, there was no significant difference in DFS between the groups with or without imatinib adjuvant treatment. (D) Among CTHRC1-positive intermediate- and high-risk GIST patients, no significant difference between the imatinib treatment group and the surgery alone group were observed.

Correlation between CTHRC1 Expression and the Efficacy of Imatinib Adjuvant Treatment

According to the NIH classification guideline, intermediate- or high-risk GIST patients require adjuvant treatment with imatinib. In this study, we analyzed the correlation between the efficacy of imatinib adjuvant treatment and CTHRC1 expression. Among 14 CTHRC1- negative intermediate- and high-risk GIST patients, there was no correlation between imatinib treatment and patient prognosis (Figure 2C). The difference in DFS between the imatinib treatment group and the surgery treatment only group was not statistically significant (P = 0.255). Notably, among 214 CTHRC1- positive intermediate- and high-risk GIST patients, the DFS rate in the imatinib treatment group were higher than that in the surgery only group within 3 years of follow-up. However, the differences in 5-year DFS rates in the two groups were not statistically significant (P = 0.313) (Figure 2D). Therefore, the expression of CTHRC1 can not predict the efficacy of imatinib adjuvant treatment in our current study.

Recombinant CTHRC1 Protein Promotes GIST Cell Migration and Invasion in a Dose-dependent Manner

To explore the biological functions of CTHRC1 as a secreted protein, CTHRC1 was recombinantly expressed in EBNA-293 cells, and further purified and verified by western blotting (Figure W2). Then, the purified recombinant CTHRC1 (rCTHRC1) protein was applied to primary GIST cells in a migration and Matrigel invasion assay. Compared to the vehicle group, GIST cell migration and invasion were significantly enhanced by rCTHRC1 protein at doses of 1 nM, 20 nM, and 50 nM (Figure 3, C and D). Moreover, the promotion of cell motility by the rCTHRC1 protein was dose-dependent. However, primary cell viability was not remarkably affected by rCTHRC1 protein based on the cell viability assay (Figure 3B). These results demonstrate that CTHRC1 is a potent pro-invasion factor that facilitates GIST cell invasion in a dose-dependent manner.

CTHRC1 Activates Wnt/PCP-Rho Signaling in Primary GIST Cells

To understand the underlying mechanism by which CTHRC1 promotes GIST cell migration and invasion, we examined the activation of the canonical Wnt pathway and the non-canonical Wnt pathway. GIST cells were transfected with a Wnt//β-catenin reporter plasmid (TCF/catenin plasmid) and negative control counterpart plasmid or non-canonical Wnt/PCP pathway reporter plasmid (ATF2 plasmid). Recombinant CTHRC1 or vehicle control was added 24 hours after transfection, and luciferase activity was determined. The results showed that Wnt/β-catenin signaling was inhibited while the noncanonical Wnt/PCP signaling was activated by rCTHRC1 protein in primary GIST cells (Figure 4, A and B). The effects of rCTHRC1 protein on Wnt signaling was blocked by a Wnt5a neutralizing antibody (Figure 4, C and D). We also verified the block effect of Wnt3a and Wnt5a neutralizing antibodies (Figure 4, E and F). We further confirmed the inhibitory effect of CTHRC1 on Wnt/β-catenin signaling using western blotting assy. The level of phosphorylated β-catenin, which indicates the degradation of β-catenin, was increased in primary GIST cells treated with rCTHRC1 (Figure 6, B and F). In addition, the level of GSK3β, which phosphorylates β-catenin on Ser-33/Ser-37/Thr-41 was increased in rCTHRC1 treated primary GIST cells (Figure 6, B and D). Therefore, we confirmed that CTHRC1 inhibits the canonical Wnt/β-catenin pathway in primary GIST cells.

Figure 4.

Figure 4

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(A) Dual-luciferase reporter assay showed that rCthrc protein inhibited Wnt/β-catenin signaling of primary GIST cells in a dose-dependent manner. The results shown are mean±SD of relative firefly/Renilla ratio. (B) Noncanonical Wnt/PCP signaling of GIST cells was activated by rCTHRC1 protein in a dose-dependent manner. (C) The inhibitory effect of rCTHRC1 on Wnt/β-catenin signaling was partially blocked by Wnt5a monoclonal neutralizing antibody. (D) The promoting effect of rCTHRC1 protein on Wnt/PCP signaling was almost blocked by Wnt5a monoclonal neutralizing antibody. (E) The promoting effect of Wnt3a on Wnt/β-catenin signaling was almost blocked by Wnt3a monoclonal neutralizing antibody. (F) The promoting effect of Wnt5a on Wnt/PCP signaling was almost blocked by Wnt5a monoclonal neutralizing antibody. (*, P < 0.05; **, P < 0.01).

Figure 6.

Figure 6

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(A) The expression of Wnt5a and the phosphorylation of JNK were examined after treatment with rCTHRC1 by western blotting. (B) The phosphorylation of GSK3β and β-catenin were examined after treatment with rCTHRC1 by western blotting. (C) Quantitative analysis of grey value for Wnt5a using ImageJ software. The relative expression of Wnt5a induced by rCTHRC1 at 0′, 5′, 10′, 30′ was compared with the grey value of Wnt5a induced by rCTHRC1 at 60′. (D) Quantitative analysis of grey value for phospho-GSK3β/total GSK3β ratio using ImageJ software. (E) Quantitative analysis of grey value for phospho-JNK/total JNK ratio using ImageJ software. (F) Quantitative analysis of grey value for phospho-β-catenin/totalβ-catenin ratio using ImageJ software. (*, P < 0.05; **, P < 0.01).

The downstream molecules of the Wnt/PCP pathway mainly include the small GTPase family, such as Rac1, RhoA and Cdc42, which play important roles in cancer cell migration and invasion. Using a Rho GTPases pull-down assay, we found that the rCTHRC1 protein enhanced the activity of RhoA and Rac1 but not Cdc42 (Figure 5, A-D).

Figure 5.

Figure 5

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(A) Analysis of the active and total RhoA,Rac1 and Cdc42 in primary GIST cells treated with rCTHRC1 protein by pull-down assay.(B) Quantitative analysis of grey value for active Rac1/ -total Rac1 ratio using ImageJ software. (C) Quantitative analysis of grey value for active RhoA/ -total RhoA ratio using ImageJ software. (D) Quantitative analysis of grey value for active Cdc42/ total Cdc42 ratio using ImageJ software. (E) Rac1 G-LISA assay was used to assess the levels of GTP-bound Rac1 in GIST cells treated with rCTHRC1 protein. (F) RhoA G-LISA assay was used to assess the levels of GTP-bound RhoA in GIST cells treated with rCTHRC1 protein. (*, P < 0.05; **, P < 0.01).

To further confirm the above results, the GLISA assay, another approach to measure the activities of Rho GTPases, was performed. It also demonstrated that the activities of RhoA and Rac1 were significantly enhanced by rCTHRC1 treatment in primary GIST cells, which is consistent with the results of the Rho GTPases pull-down assay (Figure 5, E and F).

Furthermore, the phosphorylation of c-Jun N terminal kinase (JNK), another downstream molecule of the Wnt/PCP pathway, and Wnt5a were also elevated by rCTHRC1 treatment (Figure 6, A, C and E). These results suggested that CTHRC1 may promote GIST cell invasion through the Wnt/PCP-Rho-JNK pathway.

CTHRC1-induced Primary GIST Cell Migration and Invasion Is Wnt5a and Wnt/PCP Signaling-dependent

We further investigated whether Wnt3a (a ligand of canonical Wnt/β-catenin pathway) and Wnt5a (a ligand of noncanonical Wnt/PCP pathway) are involved in CTHRC1-induced GIST cell migration and invasion by using neutralizing antibodies of Wnt3a and Wnt5a. The data illustrated that the migration- and invasion-promoting activities of rCTHRC1 at a dose of 20 nM were not affected by the Wnt3a neutralizing antibody (Figure 7, A and B, Figure 8, A and B). However, the promoting effects of rCTHRC1 on GIST cell migration and invasion were almost completely blocked by the Wnt5a neutralizing antibody (Figure 7, A and B, Figure 8, A and B). We further investigated whether the promoting effects of CTHRC1 on GIST cells motility are Wnt/PCP signaling-dependent by using inhibitors of ROCK and Rac1, which are key downstream molecules of the Wnt/PCP pathway. The results showed that both ROCK and Rac1 inhibitors treatment inhibits the promoting effects of rCTHRC1 on GIST cell migration and invasion (Figure 7, C and D, Figure 8, C and D).

Figure 7.

Figure 7

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(A) The promotive effect of rCTHRC1 protein was blocked by Wnt5a neutralizing antibody but not blocked by Wnt3a neutralizing antibody shown from cell migration assay in vitro. (B) Quantification analysis of migrated cells were performed for six randomly selected fields (original magnification:200 ×). (C) The promotive effect of rCTHRC1 protein was partially blocked by Rac1 inhibitor (NSC23766) as well as ROCK inhibitor (Y-27632) shown from cell migration assay. (D) Quantification analysis of migrated cells were performed for six randomly selected fields (original magnification:200 ×). (*, P < 0.05; **, P < 0.01).

Figure 8.

Figure 8

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(A) The pro-invasion effect of rCTHRC1 protein was blocked by Wnt5a neutralizing antibody but not blocked by Wnt3a neutralizing antibody shown from cell invasion assay in vitro. (B) Quantification analysis of migrated cells were performed for six randomly selected fields (original magnification: 200 ×). (C) The pro-invasion effect of rCTHRC1 protein was partially blocked by Rac1 inhibitor (NSC23766) as well as ROCK inhibitor (Y-27632) shown from cell invasion assay. (D) Quantification analysis of migrated cells were performed for six randomly selected fields (original magnification:200 ×). (*, P < 0.05; **, P < 0.01).

Taken together, these data indicated that the CTHRC1-induced GIST cell migration and invasion is Wnt5a and noncanonical Wnt/PCP signaling dependent (Figure 9).

Figure 9.

Figure 9

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CTHRC1 induced cell signaling alteration and its related cell movement.

Discussion

GISTs have a variable malignancy degree ranging from a curable disorder to highly malignant sarcomas [1,8]. The majority of GISTs stain positive for KIT oncoproteins in immunohistochemical assays [3,24]. KIT is a stem cell growth factor receptor that plays pro-proliferative and anti-apoptotic roles in GIST progression [5,6]. GIST patients treated with the KIT targeted inhibitor, − imatinib, showed prolonged median recurrence-free survival of 12 to 24 months [1,21]. Recurrence and metastasis in GIST patients are the major causes of treatment failure or even death [7,25,26]. Thus, new predictive biomarkers for recurrence and an understanding of the mechanisms of GIST metastasis are urgently needed.

By analyzing the GIST microarray dataset (GSE21315) from the GEO database (Figure W1A), we found that CTHRC1 expression in GIST with liver metastasis was remarkably higher than in primary GIST tissues(fold change>3, P < 0.05). This result strongly suggested that CTHRC1 may play important roles in regulating GIST metastasis. The NIH classification published in 2002 was widely accepted as standard for predicting the prognosis of GIST patients [27,28]. According to the NIH classification, the risk assessments are based on tumor size and the number of mitotic figures. We have analyzed the correlation between CTHRC1 expression and GIST clinicopathological parameters and found that the CTHRC1 expression levels were closely related to NIH classification, tumor size and the number of mitotic figures. These analyses suggest that CTHRC1-positive GISTs exhibit a greater likelihood of malignant behavior and more aggressive features. Moreover, there was a significant difference between male and female GIST patients in frequency of high CTHRC1 levels (Table 1). It has been reported that CTHRC1 is associated with attenuated inflammatory arthritis severity in males, but not in females [29,30]. The naive mice assay showed that the expression and inducibility of CTHRC1 were highly dependent on sex [29,30]. Among naive wild-type BALB/c mice, CTHRC1 expression was remarkably higher in males than in females. Moreover, CTHRC1 was one of the major sex-affected differentially expressed genes [29,30]. Therefore, sex disparities may cause the difference between male and female GIST patients in high CTHRC1 expression rates.

The Kaplan-Meier curves analysis revealed that CTHRC1 expression was closely correlated with OS and DFS of GIST patients. GIST patients with CTHRC1-positive tumors had shorter OS and DFS than CTHRC1-negative patients. Therefore, we identified that CTHRC1 is an available predictor of poor prognosis including OS and DFS in GIST patients. In addition, the great clinical value of CTHRC1 in predicting the recurrence risk of postoperative GIST patients may contribute to improving the clinical therapeutic effects.

Tumor microenvironments including components of extracellular matrix protein play crucial roles in promoting tumor invasion and metastasis [31]. CTHRC1, a secreted ECM protein, has been reported to be up-regulated in many solid tumors. In hepatocellular carcinoma, CTHRC1 is up-regulated and promotes tumor invasion and predicts poor prognosis [13]. CTHRC1 plays a promoting role in pancreatic cancer progression and metastasis by enhancing the migration ability of cancer cells [18]. These accumulating data indicate that CTHRC1 is an important regulator of tumor invasion and metastasis in the tumor microenvironment. In the present study, we have found that CTHRC1 expression in GIST tissue is gradually elevated in accordance with risk grading. Based on an in vitro functional assay, CTHRC1 was considered to be an invasion-promoting protein and ultimately contributed to gastrointestinal stromal tumor metastasis and recurrence.

Although the functional roles of CTHRC1 in tumor cell invasion and metastasis have been well established, the underlying mechanisms of how CTHRC1 promotes cancer cell invasion remains unclear. It has been reported that CTHRC1 acts as a Wnt cofactor that selectively activates the PCP pathway in the inner ear developmental process [17]. In the present study, we showed that CTHRC1 promotes GIST cell invasion by activating Wnt/PCP signaling, which is supported by the following evidence. First, the luciferase reporter assay and western blotting showed that recombinant CTHRC1 protein activated the PCP pathway of Wnt signaling of primary GIST cells in a dose-dependent manner. Second, the pro-invasion activity of the rCTHRC1 protein was blocked by the neutralizing antibody of Wnt5a (a ligand for Wnt/PCP pathway [32,33]) and the inhibitors of Rac1 and ROCK (the downstream molecules of Wnt/PCP signaling [34,35]).

CTHRC1 promoted tumor cells migration by activating Rac1 and resulted in metastasis of pancreatic cancer [18]. The overexpression of CTHRC1 promotes tumor invasion by activating RhoA in hepatocellular carcinoma [13]. Accordingly, we demonstrated the promoting effect of CTHRC1 on both RhoA and Rac1 in GIST cells. Moreover, we further verified that the pro-invasion activity of CTHRC1 in GIST cells was dependent on the Wnt5a/PCP-Rho axis by blocking the Wnt5a/PCP-Rho pathway with neutralizing antibodies and specific inhibitors. The noncanonical Wnt/PCP pathway transmits signaling from the cell-surface Frizzled receptor-coupled Wnt5a protein, via the Dvl-RhoA/Rac1-JNK-ATF2/c-Jun cascade [36-38], to the nucleus. Noncanonical Wnt/PCP signaling plays important roles in promoting cell migration [39] and formation of cell protrusions [40,41]. The small Rho GTPases Rac1, RhoA and Cdc42, are key executors of Wnt/PCP related cell migration [42,43]. RhoA controls the assembly of actin to generate contractile forces [44,45], while Rac1 and Cdc42 promote actin polymerization contributing to the formation of protrusive forces [46,47]. Therefore, the efficient cell movement requires synergistic actions of the three Rho GTPases [48-50]. In this study, we have shown that CTHRC1, a secreted protein, transduces outside-in signals through the Wnt/PCP pathway and coordinates the action of the three Rho GTPases to promote GIST cell migration and invasion.

Taken together, we have demonstrated that CTHRC1 expression level is closely correlated with risk grade of NIH classification and prognosis of GIST, indicating that CTHRC1 served as a new predictor of recurrence risk and prognosis in post-operative GIST patients. Furthermore, we have shown that CTHRC1 promotes GIST cell migration and invasion by activating the Wnt/PCP-Rho signaling, suggesting that the CTHRC1- Wnt/PCP-Rho axis may be a new therapeutic target for interventions against GIST invasion and metastasis.

Acknowledgments

The study was supported by the National Science Foundation of China (81071738; 81101600, 81272743 and 81302094), Shanghai Health Bureau Youth Fund (2009Y110). ZGZ and HC were the principal investigator and supervised the implementation of the study. MZM wrote the protocols and, with CZ, XMY, ZZZ and HYY, analyzed the data and interpreted the findings. CZ and MZM were responsible for the collection of clinical data. HM had successfully isolated primary GIST cells from patients' tissues. MZM and CZ carried out the experiments involved in this article. HC, ZGZ, JRG, SLY and WXQ participated in critical revision of the manuscript for important intellectual content. All authors had full access to the primary data and the final analysis and approved the final version of the manuscript.

These authors declare no conflict of interest.

Footnotes

1

This article refers to supplementary materials, which is designated by Table W1 and Figures W1 to W3 is available online at www.neoplasia.com.

Contributor Information

Ming-Ze Ma, Email: caohuishcn@hotmail.com.

Chun Zhuang, Email: zhuangchun88@163.com.

Xiao-Mei Yang, Email: xmyang@sibs.ac.cn.

Zi-Zhen Zhang, Email: zzzhang16@hotmail.com.

Hong Ma, Email: mahong4899@126.com.

Wen-Ming Zhang, Email: wenming_zhang@163.com.

Haiyan You, Email: bettyyouyou@yeah.net.

Wenxin Qin, Email: wxqin@sjtu.edu.cn.

Jianren Gu, Email: jrgu@shsci.org.

Shengli Yang, Email: slyang@sibs.ac.cn.

Hui Cao, Email: caohuishcn@hotmail.com.

Zhi-Gang Zhang, Email: zzhang@shsci.org.

Appendix A. Supplementary materials

Figure W1.

Figure W1

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(A) Analysis of CTHRC1 differential expression in metastatic GIST and primary GIST based on data from GEO Database (GSE21315). (B) Schematic diagram of V152 vector which was used to reconstruct CTHRC1-StrepII recombinant plasmid.

Figure W2.

Figure W2

Open in a new tab

Verification of affinity purified CTHRC1 protein by Coomassie Brilliant Blue staining and Western blotting.

Figure W3.

Figure W3

Open in a new tab

Negative controls for primary and secondary antibodies in GIST TMA immunohistochemistry staining assay (original magnification:100 ×).

Table W1.

ANOVA analysis (post-hoc testing) for statistics of figures.

Multiple Comparisons
(I) 1 = low risk,2 = intermediate risk,3 = high risk (J) 1 = low risk,2 = intermediate risk,3 = high risk Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 1A
1 2 − 1.53243* .57014 .031 − 2.9405 -.1244
3 − 1.47190* .58777 .046 − 2.9235 -.0203
2 1 1.53243* .57014 .031 .1244 2.9405
3 .06053 .62280 .995 − 1.4776 1.5986
3 1 1.47190* .58777 .046 .0203 2.9235
2 -.06053 .62280 .995 − 1.5986 1.4776
VAR00001
Tukey HSD



Multiple Comparisons
(I) concentration (J) concentration Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 3B
Day0
0 1 -.0040000 .0094810 .973 -.034361 .026361
20 -.0160000 .0094810 .389 -.046361 .014361
50 -.0013333 .0094810 .999 -.031695 .029028
1 0 .0040000 .0094810 .973 -.026361 .034361
20 -.0120000 .0094810 .607 -.042361 .018361
50 .0026667 .0094810 .992 -.027695 .033028
20 0 .0160000 .0094810 .389 -.014361 .046361
1 .0120000 .0094810 .607 -.018361 .042361
50 .0146667 .0094810 .456 -.015695 .045028
50 0 .0013333 .0094810 .999 -.029028 .031695
1 -.0026667 .0094810 .992 -.033028 .027695
20 -.0146667 .0094810 .456 -.045028 .015695
VAR00001
Tukey HSD



Multiple Comparisons
(I) concentration (J) concentration Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 3B
Day1
0 1 .0023333 .0055578 .973 -.015465 .020131
20 -.0216667* .0055578 .019 -.039465 -.003869
50 -.0230000* .0055578 .014 -.040798 -.005202
1 0 -.0023333 .0055578 .973 -.020131 .015465
20 -.0240000* .0055578 .011 -.041798 -.006202
50 -.0253333* .0055578 .008 -.043131 -.007535
20 0 .0216667* .0055578 .019 .003869 .039465
1 .0240000* .0055578 .011 .006202 .041798
50 -.0013333 .0055578 .995 -.019131 .016465
50 0 .0230000* .0055578 .014 .005202 .040798
1 .0253333* .0055578 .008 .007535 .043131
20 .0013333 .0055578 .995 -.016465 .019131
VAR00001
Tukey HSD



Multiple Comparisons
(I) concentration (J) concentration Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 3B
Day2
0 1 -.0596667* .0133083 .009 -.102285 -.017049
20 -.0253333 .0133083 .299 -.067951 .017285
50 -.0746667* .0133083 .002 -.117285 -.032049
1 0 .0596667* .0133083 .009 .017049 .102285
20 .0343333 .0133083 .120 -.008285 .076951
50 -.0150000 .0133083 .684 -.057618 .027618
20 0 .0253333 .0133083 .299 -.017285 .067951
1 -.0343333 .0133083 .120 -.076951 .008285
50 -.0493333* .0133083 .025 -.091951 -.006715
50 0 .0746667* .0133083 .002 .032049 .117285
1 .0150000 .0133083 .684 -.027618 .057618
20 .0493333* .0133083 .025 .006715 .091951
VAR00001
Tukey HSD



Multiple Comparisons
(I) concentration (J) concentration Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 3B
Day3
0 1 -.0046667 .0051962 .806 -.021307 .011973
20 -.0223333* .0051962 .011 -.038973 -.005693
50 -.0283333* .0051962 .003 -.044973 -.011693
1 0 .0046667 .0051962 .806 -.011973 .021307
20 -.0176667* .0051962 .038 -.034307 -.001027
50 -.0236667* .0051962 .008 -.040307 -.007027
20 0 .0223333* .0051962 .011 .005693 .038973
1 .0176667* .0051962 .038 .001027 .034307
50 -.0060000 .0051962 .669 -.022640 .010640
50 0 .0283333* .0051962 .003 .011693 .044973
1 .0236667* .0051962 .008 .007027 .040307
20 .0060000 .0051962 .669 -.010640 .022640
VAR00001
Tukey HSD



Multiple Comparisons
(I) concentration (J) concentration Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 3B
Day4
0 1 -.0030000 .0072111 .974 -.026092 .020092
20 .0173333 .0072111 .154 -.005759 .040426
50 -.0753333* .0072111 .000 -.098426 -.052241
1 0 .0030000 .0072111 .974 -.020092 .026092
20 .0203333 .0072111 .086 -.002759 .043426
50 -.0723333* .0072111 .000 -.095426 -.049241
20 0 -.0173333 .0072111 .154 -.040426 .005759
1 -.0203333 .0072111 .086 -.043426 .002759
50 -.0926667* .0072111 .000 -.115759 -.069574
50 0 .0753333* .0072111 .000 .052241 .098426
1 .0723333* .0072111 .000 .049241 .095426
20 .0926667* .0072111 .000 .069574 .115759
VAR00001
Tukey HSD



Multiple Comparisons
(I) concentration (J) concentration Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 3B
Day5
0 1 .0146667 .0078916 .316 -.010605 .039938
20 -.0660000* .0078916 .000 -.091272 -.040728
50 -.0893333* .0078916 .000 -.114605 -.064062
1 0 -.0146667 .0078916 .316 -.039938 .010605
20 -.0806667* .0078916 .000 -.105938 -.055395
50 -.1040000* .0078916 .000 -.129272 -.078728
20 0 .0660000* .0078916 .000 .040728 .091272
1 .0806667* .0078916 .000 .055395 .105938
50 -.0233333 .0078916 .071 -.048605 .001938
50 0 .0893333* .0078916 .000 .064062 .114605
1 .1040000* .0078916 .000 .078728 .129272
20 .0233333 .0078916 .071 -.001938 .048605
VAR00001
Tukey HSD



Multiple Comparisons
(I) concentration (J) concentration Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 3B
Day6
0 1 -.0046667 .0106797 .970 -.038867 .029533
20 -.0750000* .0106797 .001 -.109200 -.040800
50 -.1590000* .0106797 .000 -.193200 -.124800
1 0 .0046667 .0106797 .970 -.029533 .038867
20 -.0703333* .0106797 .001 -.104533 -.036133
50 -.1543333* .0106797 .000 -.188533 -.120133
20 0 .0750000* .0106797 .001 .040800 .109200
1 .0703333* .0106797 .001 .036133 .104533
50 -.0840000* .0106797 .000 -.118200 -.049800
50 0 .1590000* .0106797 .000 .124800 .193200
1 .1543333* .0106797 .000 .120133 .188533
20 .0840000* .0106797 .000 .049800 .118200
VAR00001
Tukey HSD



Multiple Comparisons
(I) VAR00002 (J) VAR00002 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 3C
0 1 − 6.50000* 1.69558 .005 − 11.2458 − 1.7542
20 − 55.50000* 1.69558 .000 − 60.2458 − 50.7542
50 − 69.16667* 1.69558 .000 − 73.9125 − 64.4208
1 0 6.50000* 1.69558 .005 1.7542 11.2458
20 − 49.00000* 1.69558 .000 − 53.7458 − 44.2542
50 − 62.66667* 1.69558 .000 − 67.4125 − 57.9208
20 0 55.50000* 1.69558 .000 50.7542 60.2458
1 49.00000* 1.69558 .000 44.2542 53.7458
50 − 13.66667* 1.69558 .000 − 18.4125 − 8.9208
50 0 69.16667* 1.69558 .000 64.4208 73.9125
1 62.66667* 1.69558 .000 57.9208 67.4125
20 13.66667* 1.69558 .000 8.9208 18.4125
VAR00001
Tukey HSD



Multiple Comparisons
(I) concentration (J) concentration Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 3D
0 1 − 23.66667* 3.28549 .000 − 32.8625 − 14.4708
20 − 55.50000* 3.28549 .000 − 64.6959 − 46.3041
50 − 72.16667* 3.28549 .000 − 81.3625 − 62.9708
1 0 23.66667* 3.28549 .000 14.4708 32.8625
20 − 31.83333* 3.28549 .000 − 41.0292 − 22.6375
50 − 48.50000* 3.28549 .000 − 57.6959 − 39.3041
20 0 55.50000* 3.28549 .000 46.3041 64.6959
1 31.83333* 3.28549 .000 22.6375 41.0292
50 − 16.66667* 3.28549 .000 − 25.8625 − 7.4708
50 0 72.16667* 3.28549 .000 62.9708 81.3625
1 48.50000* 3.28549 .000 39.3041 57.6959
20 16.66667* 3.28549 .000 7.4708 25.8625
VAR00001
Tukey HSD



Multiple Comparisons
(I) VAR00002 (J) VAR00002 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 4A (Mutant TCF)
0 1 -.01200 .02143 .941 -.0806 .0566
20 -.02933 .02143 .550 -.0980 .0393
50 -.04333 .02143 .257 -.1120 .0253
1 0 .01200 .02143 .941 -.0566 .0806
20 -.01733 .02143 .849 -.0860 .0513
50 -.03133 .02143 .500 -.1000 .0373
20 0 .02933 .02143 .550 -.0393 .0980
1 .01733 .02143 .849 -.0513 .0860
50 -.01400 .02143 .911 -.0826 .0546
50 0 .04333 .02143 .257 -.0253 .1120
1 .03133 .02143 .500 -.0373 .1000
20 .01400 .02143 .911 -.0546 .0826
VAR00001
Tukey HSD



Multiple Comparisons
(I) VAR00002 (J) VAR00002 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 4A (TCF)
0 1 .19167* .02076 .000 .1252 .2582
20 .30900* .02076 .000 .2425 .3755
50 .46600* .02076 .000 .3995 .5325
1 0 -.19167* .02076 .000 -.2582 -.1252
20 .11733* .02076 .002 .0508 .1838
50 .27433* .02076 .000 .2078 .3408
20 0 -.30900* .02076 .000 -.3755 -.2425
1 -.11733* .02076 .002 -.1838 -.0508
50 .15700* .02076 .000 .0905 .2235
50 0 -.46600* .02076 .000 -.5325 -.3995
1 -.27433* .02076 .000 -.3408 -.2078
20 -.15700* .02076 .000 -.2235 -.0905
VAR00001
Tukey HSD



Multiple Comparisons
(I) VAR00002 (J) VAR00002 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 4B
0 1 − 1.92467* .41532 .007 − 3.2547 -.5947
20 − 2.50733* .41532 .001 − 3.8373 − 1.1773
50 − 6.96000* .41532 .000 − 8.2900 − 5.6300
1 0 1.92467* .41532 .007 .5947 3.2547
20 -.58267 .41532 .531 − 1.9127 .7473
50 − 5.03533* .41532 .000 − 6.3653 − 3.7053
20 0 2.50733* .41532 .001 1.1773 3.8373
1 .58267 .41532 .531 -.7473 1.9127
50 − 4.45267* .41532 .000 − 5.7827 − 3.1227
50 0 6.96000* .41532 .000 5.6300 8.2900
1 5.03533* .41532 .000 3.7053 6.3653
20 4.45267* .41532 .000 3.1227 5.7827
VAR00001
Tukey HSD



Multiple Comparisons
(I) 1 = 0nm,2 = 20nm,3 = 0nm+Wnt5a mAb,4 = 20nm+Wnt5a mAb (J) 1 = 0nm,2 = 20nm,3 = 0nm+Wnt5a mAb,4 = 20nm+Wnt5a mAb Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 4C
1 2 .33467* .03334 .000 .2279 .4414
3 .04400 .03334 .577 -.0628 .1508
4 .16100* .03334 .006 .0542 .2678
2 1 -.33467* .03334 .000 -.4414 -.2279
3 -.29067* .03334 .000 -.3974 -.1839
4 -.17367* .03334 .004 -.2804 -.0669
3 1 -.04400 .03334 .577 -.1508 .0628
2 .29067* .03334 .000 .1839 .3974
4 .11700* .03334 .033 .0102 .2238
4 1 -.16100* .03334 .006 -.2678 -.0542
2 .17367* .03334 .004 .0669 .2804
3 -.11700* .03334 .033 -.2238 -.0102
VAR00001
Tukey HSD



Multiple Comparisons
(I) VAR00002 (J) VAR00002 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 4D
1 2 − 2.75433* .27391 .000 − 3.6315 − 1.8772
3 1.07233* .27391 .019 .1952 1.9495
4 -.32100 .27391 .659 − 1.1981 .5561
2 1 2.75433* .27391 .000 1.8772 3.6315
3 3.82667* .27391 .000 2.9495 4.7038
4 2.43333* .27391 .000 1.5562 3.3105
3 1 − 1.07233* .27391 .019 − 1.9495 -.1952
2 − 3.82667* .27391 .000 − 4.7038 − 2.9495
4 − 1.39333* .27391 .004 − 2.2705 -.5162
4 1 .32100 .27391 .659 -.5561 1.1981
2 − 2.43333* .27391 .000 − 3.3105 − 1.5562
3 1.39333* .27391 .004 .5162 2.2705
VAR00001
Tukey HSD



Multiple Comparisons
(I) 1 = NC,2 = + Wnt3a,3 = + Wnt3a mAb,4 = Wnt3a+Wnt3a mAb (J) 1 = NC,2 = + Wnt3a,3 = + Wnt3a mAb,4 = Wnt3a+Wnt3a mAb Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 4E
1 2 -.47433* .04523 .000 -.6192 -.3295
3 .17733* .04523 .019 .0325 .3222
4 .16567* .04523 .026 .0208 .3105
2 1 .47433* .04523 .000 .3295 .6192
3 .65167* .04523 .000 .5068 .7965
4 .64000* .04523 .000 .4951 .7849
3 1 -.17733* .04523 .019 -.3222 -.0325
2 -.65167* .04523 .000 -.7965 -.5068
4 -.01167 .04523 .994 -.1565 .1332
4 1 -.16567* .04523 .026 -.3105 -.0208
2 -.64000* .04523 .000 -.7849 -.4951
3 .01167 .04523 .994 -.1332 .1565
VAR00001
Tukey HSD



Multiple Comparisons
(I) 1 = NC,2 = + Wnt5a,3 = + Wnt5a mAb,4 = Wnt5a+Wnt5a mAb (J) 1 = NC,2 = + Wnt5a,3 = + Wnt5a mAb,4 = Wnt5a+Wnt5a mAb Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 4F
1 2 − 3.02767* .20239 .000 − 3.6758 − 2.3795
3 1.02867* .20239 .004 .3805 1.6768
4 -.17100 .20239 .832 -.8191 .4771
2 1 3.02767* .20239 .000 2.3795 3.6758
3 4.05633* .20239 .000 3.4082 4.7045
4 2.85667* .20239 .000 2.2085 3.5048
3 1 − 1.02867* .20239 .004 − 1.6768 -.3805
2 − 4.05633* .20239 .000 − 4.7045 − 3.4082
4 − 1.19967* .20239 .002 − 1.8478 -.5515
4 1 .17100 .20239 .832 -.4771 .8191
2 − 2.85667* .20239 .000 − 3.5048 − 2.2085
3 1.19967* .20239 .002 .5515 1.8478
VAR00001
Tukey HSD



Multiple Comparisons
(I) VAR00002 (J) VAR00002 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 5B
0 5 -.58300* .05290 .000 -.7571 -.4089
10 -.12600 .05290 .197 -.3001 .0481
30 -.40433* .05290 .000 -.5784 -.2302
60 -.13600 .05290 .150 -.3101 .0381
5 0 .58300* .05290 .000 .4089 .7571
10 .45700* .05290 .000 .2829 .6311
30 .17867* .05290 .044 .0046 .3528
60 .44700* .05290 .000 .2729 .6211
10 0 .12600 .05290 .197 -.0481 .3001
5 -.45700* .05290 .000 -.6311 -.2829
30 -.27833* .05290 .003 -.4524 -.1042
60 -.01000 .05290 1.000 -.1841 .1641
30 0 .40433* .05290 .000 .2302 .5784
5 -.17867* .05290 .044 -.3528 -.0046
10 .27833* .05290 .003 .1042 .4524
60 .26833* .05290 .003 .0942 .4424
60 0 .13600 .05290 .150 -.0381 .3101
5 -.44700* .05290 .000 -.6211 -.2729
10 .01000 .05290 1.000 -.1641 .1841
30 -.26833* .05290 .003 -.4424 -.0942
VAR00001
Tukey HSD



Multiple Comparisons
(I) VAR00002 (J) VAR00002 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 5C
0 5 -.20667* .01588 .000 -.2589 -.1544
10 -.39767* .01588 .000 -.4499 -.3454
30 -.52167* .01588 .000 -.5739 -.4694
60 -.53733* .01588 .000 -.5896 -.4851
5 0 .20667* .01588 .000 .1544 .2589
10 -.19100* .01588 .000 -.2433 -.1387
30 -.31500* .01588 .000 -.3673 -.2627
60 -.33067* .01588 .000 -.3829 -.2784
10 0 .39767* .01588 .000 .3454 .4499
5 .19100* .01588 .000 .1387 .2433
30 -.12400* .01588 .000 -.1763 -.0717
60 -.13967* .01588 .000 -.1919 -.0874
30 0 .52167* .01588 .000 .4694 .5739
5 .31500* .01588 .000 .2627 .3673
10 .12400* .01588 .000 .0717 .1763
60 -.01567 .01588 .855 -.0679 .0366
60 0 .53733* .01588 .000 .4851 .5896
5 .33067* .01588 .000 .2784 .3829
10 .13967* .01588 .000 .0874 .1919
30 .01567 .01588 .855 -.0366 .0679
VAR00001
Tukey HSD



Multiple Comparisons
(I) time (J) time Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 5D
0 5 .03000 .03961 .937 -.1004 .1604
10 -.01000 .03961 .999 -.1404 .1204
30 .00667 .03961 1.000 -.1237 .1370
60 -.02333 .03961 .974 -.1537 .1070
5 0 -.03000 .03961 .937 -.1604 .1004
10 -.04000 .03961 .845 -.1704 .0904
30 -.02333 .03961 .974 -.1537 .1070
60 -.05333 .03961 .671 -.1837 .0770
10 0 .01000 .03961 .999 -.1204 .1404
5 .04000 .03961 .845 -.0904 .1704
30 .01667 .03961 .992 -.1137 .1470
60 -.01333 .03961 .997 -.1437 .1170
30 0 -.00667 .03961 1.000 -.1370 .1237
5 .02333 .03961 .974 -.1070 .1537
10 -.01667 .03961 .992 -.1470 .1137
60 -.03000 .03961 .937 -.1604 .1004
60 0 .02333 .03961 .974 -.1070 .1537
5 .05333 .03961 .671 -.0770 .1837
10 .01333 .03961 .997 -.1170 .1437
30 .03000 .03961 .937 -.1004 .1604
VAR00001
Tukey HSD



Multiple Comparisons
(I) VAR00002 (J) VAR00002 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 5E
0 1 -.11033* .02696 .015 -.1967 -.0240
20 -.32633* .02696 .000 -.4127 -.2400
50 -.47333* .02696 .000 -.5597 -.3870
1 0 .11033* .02696 .015 .0240 .1967
20 -.21600* .02696 .000 -.3023 -.1297
50 -.36300* .02696 .000 -.4493 -.2767
20 0 .32633* .02696 .000 .2400 .4127
1 .21600* .02696 .000 .1297 .3023
50 -.14700* .02696 .003 -.2333 -.0607
50 0 .47333* .02696 .000 .3870 .5597
1 .36300* .02696 .000 .2767 .4493
20 .14700* .02696 .003 .0607 .2333
VAR00001
Tukey HSD



Multiple Comparisons
(I) VAR00002 (J) VAR00002 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 5F
0 1 -.15167* .01790 .000 -.2090 -.0943
20 -.39267* .01790 .000 -.4500 -.3353
50 -.51900* .01790 .000 -.5763 -.4617
1 0 .15167* .01790 .000 .0943 .2090
20 -.24100* .01790 .000 -.2983 -.1837
50 -.36733* .01790 .000 -.4247 -.3100
20 0 .39267* .01790 .000 .3353 .4500
1 .24100* .01790 .000 .1837 .2983
50 -.12633* .01790 .000 -.1837 -.0690
50 0 .51900* .01790 .000 .4617 .5763
1 .36733* .01790 .000 .3100 .4247
20 .12633* .01790 .000 .0690 .1837
VAR00001
Tukey HSD



Multiple Comparisons
(I) VAR00002 (J) VAR00002 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 6C
0 5 -.08967* .02012 .008 -.1559 -.0234
10 -.37167* .02012 .000 -.4379 -.3054
30 -.56933* .02012 .000 -.6356 -.5031
60 -.74133* .02012 .000 -.8076 -.6751
5 0 .08967* .02012 .008 .0234 .1559
10 -.28200* .02012 .000 -.3482 -.2158
30 -.47967* .02012 .000 -.5459 -.4134
60 -.65167* .02012 .000 -.7179 -.5854
10 0 .37167* .02012 .000 .3054 .4379
5 .28200* .02012 .000 .2158 .3482
30 -.19767* .02012 .000 -.2639 -.1314
60 -.36967* .02012 .000 -.4359 -.3034
30 0 .56933* .02012 .000 .5031 .6356
5 .47967* .02012 .000 .4134 .5459
10 .19767* .02012 .000 .1314 .2639
60 -.17200* .02012 .000 -.2382 -.1058
60 0 .74133* .02012 .000 .6751 .8076
5 .65167* .02012 .000 .5854 .7179
10 .36967* .02012 .000 .3034 .4359
30 .17200* .02012 .000 .1058 .2382
VAR00001
Tukey HSD



Multiple Comparisons
(I) time (J) time Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 6D
0 5 -.23467* .02028 .000 -.3014 -.1679
10 -.82533* .02028 .000 -.8921 -.7586
30 -.68733* .02028 .000 -.7541 -.6206
60 -.46233* .02028 .000 -.5291 -.3956
5 0 .23467* .02028 .000 .1679 .3014
10 -.59067* .02028 .000 -.6574 -.5239
30 -.45267* .02028 .000 -.5194 -.3859
60 -.22767* .02028 .000 -.2944 -.1609
10 0 .82533* .02028 .000 .7586 .8921
5 .59067* .02028 .000 .5239 .6574
30 .13800* .02028 .000 .0712 .2048
60 .36300* .02028 .000 .2962 .4298
30 0 .68733* .02028 .000 .6206 .7541
5 .45267* .02028 .000 .3859 .5194
10 -.13800* .02028 .000 -.2048 -.0712
60 .22500* .02028 .000 .1582 .2918
60 0 .46233* .02028 .000 .3956 .5291
5 .22767* .02028 .000 .1609 .2944
10 -.36300* .02028 .000 -.4298 -.2962
30 -.22500* .02028 .000 -.2918 -.1582
VAR00001
Tukey HSD



Multiple Comparisons
(I) time (J) time Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 6E
0 5 .02467 .02220 .797 -.0484 .0977
10 -.33967* .02220 .000 -.4127 -.2666
30 -.26100* .02220 .000 -.3341 -.1879
60 -.37767* .02220 .000 -.4507 -.3046
5 0 -.02467 .02220 .797 -.0977 .0484
10 -.36433* .02220 .000 -.4374 -.2913
30 -.28567* .02220 .000 -.3587 -.2126
60 -.40233* .02220 .000 -.4754 -.3293
10 0 .33967* .02220 .000 .2666 .4127
5 .36433* .02220 .000 .2913 .4374
30 .07867* .02220 .034 .0056 .1517
60 -.03800 .02220 .469 -.1111 .0351
30 0 .26100* .02220 .000 .1879 .3341
5 .28567* .02220 .000 .2126 .3587
10 -.07867* .02220 .034 -.1517 -.0056
60 -.11667* .02220 .003 -.1897 -.0436
60 0 .37767* .02220 .000 .3046 .4507
5 .40233* .02220 .000 .3293 .4754
10 .03800 .02220 .469 -.0351 .1111
30 .11667* .02220 .003 .0436 .1897
data
Tukey HSD



Multiple Comparisons
(I) time (J) time Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 6F
0 5 -.43533* .01941 .000 -.4992 -.3715
10 -.83733* .01941 .000 -.9012 -.7735
30 -.77067* .01941 .000 -.8345 -.7068
60 -.78267* .01941 .000 -.8465 -.7188
5 0 .43533* .01941 .000 .3715 .4992
10 -.40200* .01941 .000 -.4659 -.3381
30 -.33533* .01941 .000 -.3992 -.2715
60 -.34733* .01941 .000 -.4112 -.2835
10 0 .83733* .01941 .000 .7735 .9012
5 .40200* .01941 .000 .3381 .4659
30 .06667* .01941 .040 .0028 .1305
60 .05467 .01941 .104 -.0092 .1185
30 0 .77067* .01941 .000 .7068 .8345
5 .33533* .01941 .000 .2715 .3992
10 -.06667* .01941 .040 -.1305 -.0028
60 -.01200 .01941 .969 -.0759 .0519
60 0 .78267* .01941 .000 .7188 .8465
5 .34733* .01941 .000 .2835 .4112
10 -.05467 .01941 .104 -.1185 .0092
30 .01200 .01941 .969 -.0519 .0759
data
Tukey HSD



Multiple Comparisons
(I) 1 = 0nm,2 = 20nm,3 = 0nm+Wnt3a mAb,4 = 20nm+Wnt3a mAb,5 = 0nm+Wnt5a mAb,6 = 20nm+Wnt5a mAb (J) 1 = 0nm,2 = 20nm,3 = 0nm+Wnt3a mAb,4 = 20nm+Wnt3a mAb,5 = 0nm+Wnt5a mAb,6 = 20nm+Wnt5a mAb Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 7B
1 2 − 53.00000* 4.10916 .000 − 65.4984 − 40.5016
3 -.33333 4.10916 1.000 − 12.8317 12.1651
4 − 49.50000* 4.10916 .000 − 61.9984 − 37.0016
5 − 4.50000 4.10916 .879 − 16.9984 7.9984
6 − 13.33333* 4.10916 .031 − 25.8317 -.8349
2 1 53.00000* 4.10916 .000 40.5016 65.4984
3 52.66667* 4.10916 .000 40.1683 65.1651
4 3.50000 4.10916 .955 − 8.9984 15.9984
5 48.50000* 4.10916 .000 36.0016 60.9984
6 39.66667* 4.10916 .000 27.1683 52.1651
3 1 .33333 4.10916 1.000 − 12.1651 12.8317
2 − 52.66667* 4.10916 .000 − 65.1651 − 40.1683
4 − 49.16667* 4.10916 .000 − 61.6651 − 36.6683
5 − 4.16667 4.10916 .910 − 16.6651 8.3317
6 − 13.00000* 4.10916 .038 − 25.4984 -.5016
4 1 49.50000* 4.10916 .000 37.0016 61.9984
2 − 3.50000 4.10916 .955 − 15.9984 8.9984
3 49.16667* 4.10916 .000 36.6683 61.6651
5 45.00000* 4.10916 .000 32.5016 57.4984
6 36.16667* 4.10916 .000 23.6683 48.6651
5 1 4.50000 4.10916 .879 − 7.9984 16.9984
2 − 48.50000* 4.10916 .000 − 60.9984 − 36.0016
3 4.16667 4.10916 .910 − 8.3317 16.6651
4 − 45.00000* 4.10916 .000 − 57.4984 − 32.5016
6 − 8.83333 4.10916 .290 − 21.3317 3.6651
6 1 13.33333* 4.10916 .031 .8349 25.8317
2 − 39.66667* 4.10916 .000 − 52.1651 − 27.1683
3 13.00000* 4.10916 .038 .5016 25.4984
4 − 36.16667* 4.10916 .000 − 48.6651 − 23.6683
5 8.83333 4.10916 .290 − 3.6651 21.3317
VAR00001
Tukey HSD



Multiple Comparisons
(I) 1 = 0nm,2 = 20nm,3 = 0nm+NSC23766,4 = 20nm+NSC23766,5 = 0nm+Y-27632,6 = 20nm+Y-27632 (J) 1 = 0nm,2 = 20nm,3 = 0nm+NSC23766,4 = 20nm+NSC23766,5 = 0nm+Y-27632,6 = 20nm+Y-27632 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 7D
1 2 − 51.00000* 2.31541 .000 − 58.0425 − 43.9575
3 − 4.16667 2.31541 .481 − 11.2092 2.8759
4 − 15.50000* 2.31541 .000 − 22.5425 − 8.4575
5 − 7.16667* 2.31541 .044 − 14.2092 -.1241
6 − 11.00000* 2.31541 .001 − 18.0425 − 3.9575
2 1 51.00000* 2.31541 .000 43.9575 58.0425
3 46.83333* 2.31541 .000 39.7908 53.8759
4 35.50000* 2.31541 .000 28.4575 42.5425
5 43.83333* 2.31541 .000 36.7908 50.8759
6 40.00000* 2.31541 .000 32.9575 47.0425
3 1 4.16667 2.31541 .481 − 2.8759 11.2092
2 − 46.83333* 2.31541 .000 − 53.8759 − 39.7908
4 − 11.33333* 2.31541 .000 − 18.3759 − 4.2908
5 − 3.00000 2.31541 .785 − 10.0425 4.0425
6 − 6.83333 2.31541 .061 − 13.8759 .2092
4 1 15.50000* 2.31541 .000 8.4575 22.5425
2 − 35.50000* 2.31541 .000 − 42.5425 − 28.4575
3 11.33333* 2.31541 .000 4.2908 18.3759
5 8.33333* 2.31541 .013 1.2908 15.3759
6 4.50000 2.31541 .397 − 2.5425 11.5425
5 1 7.16667* 2.31541 .044 .1241 14.2092
2 − 43.83333* 2.31541 .000 − 50.8759 − 36.7908
3 3.00000 2.31541 .785 − 4.0425 10.0425
4 − 8.33333* 2.31541 .013 − 15.3759 − 1.2908
6 − 3.83333 2.31541 .570 − 10.8759 3.2092
6 1 11.00000* 2.31541 .001 3.9575 18.0425
2 − 40.00000* 2.31541 .000 − 47.0425 − 32.9575
3 6.83333 2.31541 .061 -.2092 13.8759
4 − 4.50000 2.31541 .397 − 11.5425 2.5425
5 3.83333 2.31541 .570 − 3.2092 10.8759
VAR00001
Tukey HSD



Multiple Comparisons
(I) 1 = 0nm,2 = 20nm,3 = 0nm+Wnt3a mAb,4 = 20nm+Wnt3a mAb,5 = 0nm+Wnt5a mAb,6 = 20nm+Wnt5a mAb (J) 1 = 0nm,2 = 20nm,3 = 0nm+Wnt3a mAb,4 = 20nm+Wnt3a mAb,5 = 0nm+Wnt5a mAb,6 = 20nm+Wnt5a mAb Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 8B
1 2 − 71.83333* 3.48250 .000 − 82.4257 − 61.2410
3 1.00000 3.48250 1.000 − 9.5923 11.5923
4 − 65.33333* 3.48250 .000 − 75.9257 − 54.7410
5 − 1.00000 3.48250 1.000 − 11.5923 9.5923
6 − 9.33333 3.48250 .109 − 19.9257 1.2590
2 1 71.83333* 3.48250 .000 61.2410 82.4257
3 72.83333* 3.48250 .000 62.2410 83.4257
4 6.50000 3.48250 .441 − 4.0923 17.0923
5 70.83333* 3.48250 .000 60.2410 81.4257
6 62.50000* 3.48250 .000 51.9077 73.0923
3 1 − 1.00000 3.48250 1.000 − 11.5923 9.5923
2 − 72.83333* 3.48250 .000 − 83.4257 − 62.2410
4 − 66.33333* 3.48250 .000 − 76.9257 − 55.7410
5 − 2.00000 3.48250 .992 − 12.5923 8.5923
6 − 10.33333 3.48250 .059 − 20.9257 .2590
4 1 65.33333* 3.48250 .000 54.7410 75.9257
2 − 6.50000 3.48250 .441 − 17.0923 4.0923
3 66.33333* 3.48250 .000 55.7410 76.9257
5 64.33333* 3.48250 .000 53.7410 74.9257
6 56.00000* 3.48250 .000 45.4077 66.5923
5 1 1.00000 3.48250 1.000 − 9.5923 11.5923
2 − 70.83333* 3.48250 .000 − 81.4257 − 60.2410
3 2.00000 3.48250 .992 − 8.5923 12.5923
4 − 64.33333* 3.48250 .000 − 74.9257 − 53.7410
6 − 8.33333 3.48250 .191 − 18.9257 2.2590
6 1 9.33333 3.48250 .109 − 1.2590 19.9257
2 − 62.50000* 3.48250 .000 − 73.0923 − 51.9077
3 10.33333 3.48250 .059 -.2590 20.9257
4 − 56.00000* 3.48250 .000 − 66.5923 − 45.4077
5 8.33333 3.48250 .191 − 2.2590 18.9257
VAR00001
Tukey HSD



Multiple Comparisons
(I) 1 = 0nm,2 = 20nm,3 = 0nm+NSC23766,4 = 20nm+NSC23766,5 = 0nm+Y-27632,6 = 20nm+Y-27632 (J) 1 = 0nm,2 = 20nm,3 = 0nm+NSC23766,4 = 20nm+NSC23766,5 = 0nm+Y-27632,6 = 20nm+Y-27632 Mean Difference (I-J) Std. Error Sig. 95% Confidence Interval
Lower Bound Upper Bound
Figure 8D
1 2 − 78.00000* 3.68053 .000 − 89.1947 − 66.8053
3 − 4.16667 3.68053 .864 − 15.3613 7.0280
4 − 5.50000 3.68053 .670 − 16.6947 5.6947
5 3.16667 3.68053 .953 − 8.0280 14.3613
6 − 11.00000 3.68053 .056 − 22.1947 .1947
2 1 78.00000* 3.68053 .000 66.8053 89.1947
3 73.83333* 3.68053 .000 62.6387 85.0280
4 72.50000* 3.68053 .000 61.3053 83.6947
5 81.16667* 3.68053 .000 69.9720 92.3613
6 67.00000* 3.68053 .000 55.8053 78.1947
3 1 4.16667 3.68053 .864 − 7.0280 15.3613
2 − 73.83333* 3.68053 .000 − 85.0280 − 62.6387
4 − 1.33333 3.68053 .999 − 12.5280 9.8613
5 7.33333 3.68053 .370 − 3.8613 18.5280
6 − 6.83333 3.68053 .447 − 18.0280 4.3613
4 1 5.50000 3.68053 .670 − 5.6947 16.6947
2 − 72.50000* 3.68053 .000 − 83.6947 − 61.3053
3 1.33333 3.68053 .999 − 9.8613 12.5280
5 8.66667 3.68053 .205 − 2.5280 19.8613
6 − 5.50000 3.68053 .670 − 16.6947 5.6947
5 1 − 3.16667 3.68053 .953 − 14.3613 8.0280
2 − 81.16667* 3.68053 .000 − 92.3613 − 69.9720
3 − 7.33333 3.68053 .370 − 18.5280 3.8613
4 − 8.66667 3.68053 .205 − 19.8613 2.5280
6 − 14.16667* 3.68053 .007 − 25.3613 − 2.9720
6 1 11.00000 3.68053 .056 -.1947 22.1947
2 − 67.00000* 3.68053 .000 − 78.1947 − 55.8053
3 6.83333 3.68053 .447 − 4.3613 18.0280
4 5.50000 3.68053 .670 − 5.6947 16.6947
5 14.16667* 3.68053 .007 2.9720 25.3613
VAR00001
Tukey HSD
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⁎

The mean difference is significant at the 0.05 level.

References

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