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. 2017 Sep 15;8(49):84714–84728. doi: 10.18632/oncotarget.20950

Figure 5. TUSC3 enhances CRC cell death and is associated with prognosis of CRC patients.

Figure 5

A., Growth kinetics. SW480 cells were transfected with TUSC3 or EV plasmid, and proliferation was measured by colorimetric MTT assay after 1 to 7 days. O.D. values were calculated as -fold ± S.E. compared with day 0 (n.s., Two-way ANOVA, n = 3). B., Chemoresistance response. SW480 were transfected for 24 h as in A, followed by an incubation with 5-FU (at 50 µM), and cell viability was determined after 48 h. Data were calculated as in A compared with vehicle control (*p < 0.05 EV vs. TUSC3, Two-way ANOVA, n = 3). C.-D., TUSC3 promotes cell death (apoptosis). SW480 cells were transfected with TUSC3 or EV and incubated for 1 to 6 days. Color code: red = FLAG-TUSC3; green = actin (phalloidin); blue = nuclei (DAPI); magnification x630. Note the spread-out morphology of live TUSC3-negative cells in green vs. round dying TUSC3-positive cells in red. Representative immunofluorescence images (C) and quantitative Western blot analyses (D) from TCL (all 3 cell lines) are shown which detect cleaved PARP (apoptosis marker). Data are -fold ± S.E (*p < 0.05 EV vs. TUSC3, Two-way ANOVA, n = 3). E., TUSC3 is lost in a subset of CRCs. Tissue microarrays (TMAs) with tumor specimens from CRC patients (n = 306) were stained by immunohistochemistry (IHC). Representative images: (a) TUSC3 negative tumor cells [score 0] with black arrow marking TUSC3+ blood-derived plasma cells (positive control); (b-d) TUSC3+ tumor cells [(b) score 1, (c) score 2, (d) score 3] and stroma. F., Kaplan-Meier-Survival-Analysis. TUSC3 protein expression and its correlation to prognosis (Supplementary Table S1-S3) and clinical factors (Table S4) was calculated. Combined scores for the intensity and frequency of TUSC3 staining in tumor and stroma tissue are expressed as: 0 = negative (0-25 %), 1+ = weak positive (25-50%), 2+ = moderate positive (50-75 %), 3+ = strong positive (75-100 %). Node negative (N0) patients with low (scores 0-1) TUSC3 expression have a reduced overall survival (OS) probability compared with patients showing high (scores 2-3) TUSC3 expression (n = 135, *p = 0.035, log rank test).