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Published in final edited form as: Cell Cycle. 2009 Nov 14;8(22):3770–3776. doi: 10.4161/cc.8.22.10066

Anosmin-1 involved in neuronal cell migration is hypoxia inducible and cancer regulated

Bixi Jian 1, Chandrasekharam N Nagineni 2, Sreelatha Meleth 3, William Grizzle 4, Kirby Bland 1, Irshad Chaudry 1,5, Raghavan Raju 1,5,*
PMCID: PMC11998029  NIHMSID: NIHMS2072962  PMID: 19844165

Abstract

Functional expression of KAL1 gene is critical in the migration of GnRH neurons from the olfactory placode to the hypothalamus in embryogenesis. This gene thus far has not been shown to play a functional role in any other physiological or pathological process either in the developed brain or in peripheral tissues. We show here that KAL1 gene expression is decreased in early stage and increased in later stages of cancers. Screening of colon, lung and ovarian cancer cDNA panels indicated significant decrease in KAL1 expression in comparison to corresponding uninvolved tissues. However, KAL1 expression increased with the progression of cancer from early (I and II) stages to later (III and IV) stages of the cancer. There was a direct correlation between the TGFβ and KAL1 expression in colon cancer cDNA. Using colon cancer cell lines, we showed that TGFβ induces KAL1 gene expression and secretion of anosmin-1 protein (KAL1 coded protein). We further report that hypoxia induces anosmin-1 expression; anosmin-1 protects cancer cells from apoptosis activated by hypoxia and increases cancer cell mobility. Using siRNA technique we found that KAL1 expression following hypoxia is hypoxia-inducible factor (HIF-1)α dependent. Our results suggest that KAL1 gene expression plays an important role in cancer metastasis and protection from apoptosis.

Keywords: HIF-1α, anosmin, hypogonadism, colon cancer, apoptosis, tumor, metastasis, anosmia

Introduction

The protein, anosmin-1 encoded by KAL1 gene, is critical in facilitating the migration of GnRH neurons from the olfactory placode to the hypothalamus during organogenesis and it influences the development of both the primary and secondary olfactory processing regions.1 Kallmann Syndrome-1 (KAL1) gene defect is reported to cause anosmia (lack of smell) and hypogonadism and the disorder is known as Kallmann syndrome. As GnRH neurons that are critical in the hypothalamic-pituitary-gonadal axis and reproductive system development originate in the olfactory system, sex and smell are linked in primate development.1 Anosmin-1 is an extracellular matrix protein with adhesion and chemoattractant characteristics and contains a WAP domain and three FnIII domains.2–4 Though anosmin-1 has been implicated as a co-ligand in FGF-heparan sulphate-FGFR1 complex and the respective downstream signaling pathways, the exact mechanism of its involvement in cell adhesion and neurite outgrowth is not known.4,5

KAL1 located on the X-chromosome, has not been shown to have functional significance either in the developed brain or in peripheral tissues. Immunohistochemical analysis demonstrated the presence of anosmin-1 protein in many cells particularly epithelial cells and their basement membranes. However, the role of KAL1 gene in normal and/or pathological processes in these tissues is not known.6 Nevertheless, a microarray experiment performed using muscle biopsies of untreated and IVIg-treated dermatomyositis patients showed an upregulation of this gene in the disease and its normalization after treatment.7 While studying the regulation of expression of this gene, we found that KAL1 gene expression is significantly elevated in response to TGFβ in in vitro cultures of skeletal muscle cells.7

Among the three TGFβ isoforms expressed in mammalian epithelium, TGFβ1 is the most abundant and ubiquitously expressed isoform.8 TGFβ has long been known to be associated with cell proliferation, differentiation, migration, apoptosis as well as cancer pathogenesis and progression.9–11 Colorectal cancer is the third most common cancer and the second leading cause of cancer death in the US.12 Though TGFβ serves as a tumor suppressor in the normal intestinal epithelium by inhibiting cell proliferation and inducing apoptosis, there exists a strong correlation between the degree of differentiation of colon carcinoma cell lines and sensitivity to the antiproliferative and differentiation-promoting effects of TGFβ.8,13 In this report we sought to examine the relationship between TGFβ and KAL1 gene expression and identified for the first time that KAL1 is a hypoxia-responsive gene with a direct relationship between its expression and cancer progression.

Results

KAL1 expression in cancer tissues.

We conducted a pilot study using a cancer survey panel cDNA array representing breast, colon, kidney, liver, lung, ovary, prostate and thyroid cancers (data not shown). Three uninvolved and nine cancer tissues were in each disease group and we found that colon, lung and ovarian cancers exhibited most significant decrease in the expression of KAL1 gene. Nevertheless, there was a marked decrease in KAL1 expression in several of the tissues from the other five cancers. We therefore obtained panels with a larger number of cancer and normal specimens from colon, lung and ovarian cancers and found that the cancer tissues expressed significantly lower levels of KAL1 compared to normal controls (Fig. 1A–C).

Figure 1.

Figure 1.

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Expression of KAL1 in cancer tissues. Taqman-real time PCR analysis of KAL1 and GAPDH was performed with cancer cDNA panels representing tissues from 40–43 cancer cases and five to eight uninvolved tissues of colon (A), lung (B) and ovary (C). KAL1 mRNA levels were expressed as units relative to corresponding GAPDH levels in each panel. Inset in each panel presents bar graphs of means ± SE of uninvolved and tumor samples.

Relationship between TGFβ and KAL1 expression in colon cancer tissues.

Considering the reported inverse relationship of colon cancer to TGFβ and our results indicating a stimulatory effect of TGFβ on KAL1 gene expression in some cell types,7 we tested expression of TGFβ gene in the colon cancer cDNA panel by real time PCR. There was a direct correlation between TGFβ expression and KAL1 (Fig. 2A). The association between TGFβ and KAL1 expression in cancer patients was examined using the non-parametric spearman rank correlation coefficient. The estimated value of the correlation was 0.484 which was statistically significant (p = 0.016). However, it is not clear whether increased expression of TGFβ is responsible for increased expression of KAL1 or KAL1 is induced independent of TGFβ.

Figure 2.

Figure 2.

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TGFβ and KAL1 expression. (A) Correlation of KAL1 and TGFβ1 expression in colon cancer. Taqman real-time PCR analysis of KAL1, TGFβ and GAPDH was performed on colon cancer cDNA panels. KAL1 and TGFβ1 levels in each sample were expressed as units relative to GAPDH. Patient samples were sorted in ascending order of KAL1 expression and represented on X-axis. Each KAL1-TGFβ expression pair represents one patient. p = 0.016 by Spearman correlation. (B) TGFβ induces KAL1 gene expression in colon cancer cells. CCL-244 and Caco2 cells were treated with TGFβ (1 ng/ml) for 8 hours in serum-free medium, RNA isolated, cDNA synthesized and real-time PCR performed using KAL1 and GAPDH primer-probe pairs. Results are from representative experiments. (C) TGFβ induces anosmin-1 protein secretion. CCL-244 and Caco2 cells were treated with TGFβ for 24 hours and heparin was added 30 min prior to termination of the culture. The culture supernatant was concentrated 10 times using amicon filters (10,000 MW cut-off; Millipore, MA) and western blot carried out with anti-anosmin-1 polyclonal antibody.

TGFβ1 induces KAL1 mRNA and protein secretion in colon cancer cells.

When two colon cancer cell lines, CCL-244 and Caco-2, were treated with TGFβ we found a significant increase in KAL1 gene and anosmin-1 protein expression (Fig. 2B and C). The mRNA expression was quantified by real time PCR after treatment with TGFβ for 8 hours and the protein expression was quantified in the culture supernatant of the cells treated with TGFβ for 24 hours. When the cells cultured with TGFβ were further treated with heparin for 30 minutes prior to harvesting, a higher amount of anosmin-1 was detected in the culture supernatant (Fig. 2C, last lane) demonstrating heparin-mediated disruption of the cell surface bound anosmin-1. These data indicate that in cancer cells TGFβ significantly upregulates KAL1 mRNA and stimulates secretion of anosmin-1 protein. The secreted anosmin-1 is bound to heparin associated with extracellular matrix as reported in other cells.16

Hypoxia induces anosmin-1 in colon cancer cells.

Metastasis is most often characterized by hypoxia in the core of the primary tumor. Since KAL1 expression increased in the later stages of cancer compared to initial stages, we examined the promoter region, sequences upstream of 5’ end, of KAL1 gene for the presence of hypoxia responsive elements (HRE).17 We found a number of HRE sequences in KAL1 gene within 1,000 nucleotides upstream of the translation start site (Fig. 3A). Therefore, we conducted experiments to check whether hypoxia can induce anosmin-1, the protein product of KAL1. The colon cancer cell lines CCL-244 and Caco-2 were cultured in serum-free medium for one hour and incubated in an incubator at 2% oxygen or normoxia for 24 hours. Increased levels of anosmin-1 were detected in the supernatants of the cultures exposed to hypoxia compared to that of the cells incubated under normal oxygen conditions (Fig. 3B). These results indicate that hypoxia enhances the expression of anosmin-1 in colon cancer cells.

Figure 3.

Figure 3.

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Hypoxia and KAL1 expression. (A) KAL1 gene promoter contains hypoxia responsive elements (HRE). DNA sequence upstream of KAL1 gene coding region was searched for HRE and found to have five HRE sequences. (B) Hypoxia promotes KAL1 secretion by colon cancer cells. CCL-244 and Caco-2 cells were incubated in 2% oxygen (Innova CO-48, New Brunswick, NJ) or normoxia for 24 hr. Culture supernatants were concentrated and anosmin-1 detected by western blot using anti-anosmin polyclonal antibody. (C) Hypoxia induced anosmin-1 secretion was inhibited by HIF-1α siRNA. CCL-244 cells were transfected with siRNA to HIF-1α or siRNA to non-specific sequences and incubated for 48 hr. Then cultures were incubated in 2% oxygen for 24 hr, supernatant harvested and anosmin-1 detected by western blot. Lane 1: No hypoxia, no transfection; lane 2: No transfection, hypoxia alone; lane 3: HIF-1α siRNA from ABI and hypoxia, lane 4: HIF-1α siRNA from Dharmacon and hypoxia, lane 5: negative control siRNA from ABI and hypoxia; lane 6: neg control siRNA from Dharmacon and hypoxia.

Hypoxia-induced expression of anosmin-1 is regulated by HIF-1α.

We used siRNA to block HIF-1α to understand whether HIF-1α is involved in hypoxia-induced KAL1-1 expression in colon cancer cells. CCL-244 cells pre-treated with siRNA to HIF-1α for 48 h were subjected to hypoxia for 24 h. Hypoxia-induced anosmin-1 protein secretion was almost completely abolished by HIF-1α siRNA (Fig. 3C). There was a noticeable decrease in anosmin-1 expression in cells treated with non-specific siRNA and this was likely a non-specific effect of transfection.

KAL-1 expression is cancer stage-dependent in colon cancers.

When colon cancer cDNA panels were separated based on stage of the cancer, it was found that though the KAL1 expression decreased markedly in early stage, its expression was increased with increasing stage of the cancer. Further, we analyzed a panel of cDNAs of specimens from 24 colon cancer patients at stages 1 to 4. When we grouped them into I and II (early) stages, and III and IV (later) stages, we found increased expression of KAL1 in specimens collected at later stages of cancer (Fig. 4) compared to the early stages.

Figure 4.

Figure 4.

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Colon cancer tumor stage and KAL1 expression. Colon cancer cDNA panels derived from stage I and stage II (n = 12) and stage III and stage IV cases (n = 12) were used for determination of KAL1 expression relative to GAPDH expression levels. Significance was calculated by non-parametric Mann-Whitney test (Graph Pad Prism, CA). Bars indicate means ± SEM.

Role of anosmin-1 in cancer cell migration.

In colon cancer cDNA panels, there is increased expression of KAL1 gene with the advancing stages of cancer (Fig. 4). We examined whether anosmin-1 can facilitate the migration of cancer cells, a process associated with metastasis. CCL-244 cells pretreated with anosmin-1 or medium were placed in Boyden chamber with BSA-coated filters and incubated for 24 hr. We found a dose-dependent increase in the number of cells migrated into the membrane and we also found an increased number of cells in the lower chamber demonstrating facilitated migration of the cells by anosmin-1 (Fig. 5).

Figure 5.

Figure 5.

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Anosmin-1 induces cancer cell migration. The effect of anosmin-1 on cancer cell migration was tested using a Boyden Chamber (Haptotaxic cell migration kit, Millipore) with BSA-coated 8-micron barrier filters as per the manufacturer’s protocol. 500 μl serum-free medium was added to the lower chamber and 300 μl of a cell suspension (CCL-244) containing 1 × 106 cells in serum-free medium was added to the upper chamber. The cells pre-incubated for 30 minutes with 0, 0.5 or 2.5 μg/ml purified anosmin-1 were plated in duplicate. After overnight incubation, the chambers were removed and the cells that entered the filters (A) were stained, extracted and quantified by spectrometry as suggested by the manufacturer. The cells that migrated in to the lower chamber (B) were also extracted and quantified similarly.

Anosmin-1 acts as anti-apoptotic agent to cells exposed to hypoxia.

Our results presented above demonstrated an increase in anosmin-1 expression with advancing stages of cancer (Fig. 4) and following hypoxia (Fig. 3). However, it was not clear whether the anosmin-1 released from the cells is consequential to the apoptotic process, playing a role in facilitating apoptosis, or imparting resistance to apoptosis. In order to understand this, we pretreated colon cancer cell line CCL-244 with anosmin-1 and incubated under hypoxic condition for 24 h (Fig. 6). The protein extracts from the hypoxic cells demonstrated elevated expression of endoplasmic reticulum stress proteins ATF6 (50 kD cleavage product) and Bip, and proapoptotic proteins, caspase-3 (17 kD cleavage product) and CHOP. However the protein extracts from hypoxic cells pre-treated with anosmin-1 showed decreased levels of ATF6, Bip, caspase-3 and CHOP expression as compared to untreated cells, demonstrating anti-apoptotic effect of anosmin-1 in hypoxic cells (Fig. 6).

Figure 6.

Figure 6.

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Anosmin-1 protects colon cancer cells from hypoxia-induced apoptosis. CCL-244 cells were pre-treated with varying concentrations of affinity purified anosmin-1 or media for 30 minutes and incubated in 2% oxygen (hypoxia) or normoxia for 24 hr. The cell extracts were used for western blotting using antibodies to 50 kD cleaved fragment of ATF-6 (Abcam, MA), Bip, caspase-3, CHOP and β-actin (Cellsignalling, CA). The result shown is of one representative experiment.

Discussion

Anosmin-1 has been reported to be important in the migration of olfactory neurons and was also reported to have adhesion characteristics.1,3,18 Further our preliminary experiments demonstrated that KAL1 gene expression can be augmented by TGFβ treatment in some cell lines. Considering these characteristics we tested the expression of KAL1 in a panel of cancer specimens and found that KAL1 gene expression was decreased in most of the lung, ovary and colon cancers tested, when compared to the respective uninvolved tissues. Further we have observed that KAL1 gene and its protein product are inducible by TGFβ in both CCL-244 and Caco-2 cells, and KAL1 expression correlated with TGFβ expression in most of the cancers tested.

It is not clear whether KAL1 is a tumor suppressor gene or rather if its decreased expression is consequential to dedifferentiation of the normal epithelium to a tumor tissue. The expression of KAL1 observed in all stages of cancer did not exceed its original expression levels in normal tissues. Therefore we cannot exclude that KAL1 is acting initially as a tumor suppressor gene in the development of early cancer. The increased expression of KAL1 in late stage cancer, its responsiveness to hypoxia and augmentation of cell migration might also be important subsequently in metastasis of cancer. We are currently studying whether depletion of anosmin-1 protein in vivo (e.g., administration of antibodies to anosmin-1) can check the progression of cancer. It is suggested that with increased stage of the cancer, hypoxia sets in and hypoxic cells become metastatic.17,19,20 Hypoxia must be inducing the expression of factors that trigger metastasis.21,22 Therefore we tested the expression level of anosmin-1 in hypoxic condition and found a markedly high level of anosmin-1 in the culture supernatant of CCL-244 or Caco-2 cells exposed to 2% hypoxia. We inferred that the increased expression of anosmin-1 must be either to facilitate apoptotic cell death or to rescue the cells from apoptosis. When the colon cancer cells were pretreated with affinity purified anosmin-1 and subjected to hypoxia, the cells were protected from apoptotic process. This result indicates that anosmin-1 expression has a protective effect and therefore the survival and migratory effect of hypoxic cells might be due to enhanced expression of anosmin-1 protein. The result of the cell migration experiment using Boyden chamber substantiates the possibility that augmentation of anosmin-1 expression following hypoxia may impart migration potential to the hypoxic cells. This observation is consistent with the already reported functional role for anosmin-1 in embryogenesis, in which anosmin-1 enables the migration of GnRH neurons to the hypothalamus.

It has been suggested that KAL1 acts as a co-ligand in FGF-FGF receptor interaction.5,23 It is possible that KAL1 may act either by enhancing the anti-apoptotic effect of FGF or by acting independent of FGF-FGFR interaction, though no KAL1 receptor has been discovered so far. Our studies using neuronal cells also demonstrate a protective effect of KAL1 against hypoxic injury similar to that observed for colon cancer cell lines (data not shown). All these observations affirm a protective role for KAL1 in hypoxic condition.

KAL1 expression is regulated by multiple pathways such as that initiated by TGFβ and hypoxia. Though there is no evidence, we can not rule out the possibility that the effects of KAL1 could be mediated by TGFβ. However, considering our observation that TGFβ induces KAL1, it is likely that the observed effects are due to KAL1 or a combination of KAL1 and TGFβ. This is further supported by our discovery of HRE elements upstream of KAL1 gene, which may be regulated by HIF-1α. TGFβ is reported to act on HIF-1α accumulation and activity by increasing HIF-1α protein stability.24 Additionally the TGFβ converting enzyme, furin, is regulated by HIF-1α, a further upstream event. Therefore, the mechanism is complex and more studies are required to dissect the interdependence of TGFβ and anosmin-1 in molecular pathways that govern the development and progression of cancer including metastasis.

Decreased cellular oxygen results in decreased breakdown and stable level of HIF-1α. This allows the dimerization of HIF-1α with HIF-1β and binding of the dimer to hypoxia response element (HRE) to recruit transcriptional co-activators.25,26 We have observed the presence of five consensus HRE sequence (RCGTG) segments within 1,000 nucleotides upstream of the translation start site for KAL1 (Fig. 3A). Further, siRNA mediated disruption of HIF-1α resulted in decreased expression of anosmin-1 following hypoxia, indicating that hypoxia-induced HIF-mediated transcriptional regulation is a critical regulatory factor in KAL1 induction.

Our serendipitous finding that KAL1 gene expression is significantly decreased in the early stage of some cancers such as colon, lung and ovary may have relevance to the reported cell adhesion characteristics of anosmin-1. The observation that KAL1 gene is expressed in tissues other than the nervous system and is inducible by TGFβ also point towards its functional significance in the periphery. Our finding that KAL1 expression was significantly increased with metastasis led us to the speculation that hypoxic conditions may trigger upregulation of KAL1 expression. It has been convincingly shown that the majority of solid tumors contain regions that are poorly oxygenated, hypoxia causes both resistance to radiation and chemotherapy, and there is an association between hypoxia and metastasis in primary tumors.27–30 The association of increased prevalence of metastasis with high levels of hypoxia and the increased expression of KAL1 in metastatic tissues give credence to the significance of KAL1 in some interrelated biological functions in neuronal migration, metastasis, hypoxia, injury and gender dimorphism. Unfortunately no orthologue for KAL1 has been identified in the mouse or the rat. Nevertheless, studies using cancer cells introduced into immune-deficient mice may be a good model to facilitate further understanding of the role of KAL1 in cancer cell migration and tissue invasion.

Materials and Methods

Cells.

Colon cancer cell lines CCL-244 and Caco-2 were obtained from ATCC (Manassas, VA). HRPE (ARPE-19) cells were originally obtained from ATCC and propagated in our laboratory.14 The cells were cultured in MEM supplemented with 10% fetal bovine serum and antibiotics. To study mRNA expression, the cells were cultured to 70–80% confluence in 24-well plates in the presence or absence of TGFβ for 8 hours in serum-free medium and the cells harvested. To study protein expression, the cells were cultured either in 24-well plates or T25 flasks in the presence or absence of TGFβ (1 ng/ml) for 24 hours and incubated with or without heparin (1 μg/ml) for half an hour prior to termination of the culture.

Antibodies.

Polyclonal antibodies to human anosmin-1 were generated by immunizing rabbits with the carboxyterminal peptide CSHLKHRHPHHYKPSPERY with an added N-terminal cysteine.3 The peptides were synthesized at the peptide core in the Department of Medicine, University of Alabama at Birmingham, and the rabbit antibodies were custom produced at Brown Research (Odenville, AL). Primary antibodies to ATF-6 (Abcam, Cambridge, MA), CHOP, Bip and Caspase-3 (Cell Signalling, Danvers, MA) were used at dilutions suggested by the respective manufacturers. Secondary antibodies conjugated to peroxidase were purchased from Santa Cruz laboratory, Santa Cruz, CA.

cDNA arrays.

Custom cDNA arrays of nine different cancer types (cancer survey panel) were purchased from Origene (Rockville, MD). Three controls and seven cancer specimens were represented for each cancer type. Cancer cDNA panels representing larger number of specimens (five to eight controls and 40–43 cancer cases each) of colon, lung and ovary were also purchased from Origene.

Real-time PCR.

Real-time PCR was carried out using FAM-labeled ABI Taqman primer probe pairs for KAL1, TGFβ and GAPDH mRNA. GAPDH was used as a normalization control in each experiment. In the experiments using cells, RNA was isolated, cDNA synthesized and cDNA equivalent to 2 ng starting RNA was used for each PCR. In the experiments using cancer PCR arrays, cDNA from each patient coated onto the plates supplied by Origene were used as templates. Each reaction was carried out in 20 μl reaction volume and the PCR was performed using ABI 7500 PCR instrument. Results were calculated and expressed in fold differences relative to GAPDH expression, to normalize the amount of cDNA in each reaction.

Western blot.

Protein expression of anosmin-1 was analyzed by western blot. Briefly, total proteins in cell lysates were resolved on a 4–12% Nupage gel (Invitrogen, Carlsbad, CA) and transferred to PVDF membranes. The membranes were saturated with blocking buffer (10 mM Tris, 150 mM NaCl, and 0.05% Tween-20 supplemented with 5% dry milk) for 1 h at room temperature and incubated with polyclonal antibodies generated against anosmin-1. The membranes were then washed five times with TBST (Tris-buffered saline supplemented with 0.05% Tween-20) followed by incubation with an appropriate secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) conjugated with horseradish peroxidase for 1 h at room temperature. The membranes were again washed five times with TBST and probed using ECL (Amersham, Piscataway, NJ), and autoradiographed.15

Affinity purification of anosmin-1.

Anosmin-1 was affinity purified using protein-A sepharose immunoaffinity matrix (Pierce Biotechnology, Rockford, IL). The affinity matrix was made by cross-linking the anosmin-1 antibody to the sepharose beads using bifunctional reagent, disuccinimidyl suberate (DSS). In order to produce sufficient quantity of anosmin-1 protein, human retinal pigment epithelial (HRPE) cells were stimulated with TGFβ (1 ng/ml) overnight, and the culture supernatant was concentrated using Amicon filter (10,000 MW; Millipore). The concentrate was incubated with the affinity matrix and the bound anosmin-1 protein eluted by low pH buffer which was neutralized immediately.

Hypoxia.

Hypoxia was induced in colon cancer cell line CCL-244 by incubating at 2% oxygen in serum-free medium in an incubator (Innova, CO-48; New Brunswick Scientific, Edison, NJ) for 24 hrs. Following the hypoxic insult, the culture supernatant was saved and the cells were lysed with protein lysis buffer (Sigma, St. Louis, MO), aliquoted and frozen.

HIF-1α knockout by siRNA.

CCL-244 cells were transfected with siRNA to HIF-1α or siRNA to non-specific sequences (ABI, Foster City, CA and Dharmacon, Chicago, IL). 48 hrs after the transfection, the cells were incubated in 2% oxygen for 24 hrs, supernatant harvested and anosmin-1 detected by western blot.

Cell migration.

The effect of anosmin-1 on cancer cell migration was tested using a Boyden Chamber (Haptotaxic cell migration kit, Millipore, Billerica, MA) with BSA or collagen coated 8-micron barrier filters as per the manufacturer’s protocol. Briefly, 500 μl serum-free medium was added to the lower chamber and 300 μl of a cell suspension (CCL-244) containing 1 × 106 cells in serum-free medium was added to the upper chamber. The cells were pre-incubated for 10 minutes with 0, 0.5 or 2.5 μg purified anosmin-1 and were plated in duplicate. The chamber filters in one plate were coated with BSA while the other one was coated with collagen. After overnight incubation, the chambers were removed and the cells that entered the filters were stained, extracted and quantified by spectrometry as prescribed by the manufacturer. The cells that migrated in to the lower chamber were also extracted and quantified similarly.

Acknowledgements

This work was supported by the HSF-GEF Scholar Award.

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