Figure 2

Alterations of tumor-associated phenotypes in AGR2-silenced SNU-478 cells. A. Expression of AGR2 mRNA in SNU-478:KD (AGR2 knockdown) clones was compared with that in SNU-478:VEC (vector control) by real-time RT-PCR. The AGR2 transcript level was normalized against that of GAPDH. Results shown are means ± standard deviations of (1/2)^{CT of target - CT of GAPDH} of three experiments. B. AGR2 protein expression in SNU-478:VEC and SNU-478:KDs was detected by western blot analysis with β-actin as a loading control. The result shown is a representative of three experiments. C. Viability of SNU-478:VEC and SNU-478:KDs was measured by the MTT assay. Results shown are means ± standard deviations of three experiments (ANOVA P = 0.010; post hoc analysis by Bonferroni testing: KD2 vs. VEC, P = 0.016). D. Anchorage-independent growth was examined by colony formation assay in soft agar. The number of colonies formed in the soft agar assay with SNU-478:VEC and SNU-478:KDs. Results shown are means ± standard deviations of three experiments (ANOVA P = 0.000; post hoc analysis by Bonferroni testing: KD1-3 vs. VEC, P = 0.000). E. The number of invading cells in the transwell invasion assay with SNU-478:VEC and SNU-478:KDs. Results shown are means ± standard deviations of three experiments (ANOVA P = 0.042). F. Drug sensitivity of SNU-478:VEC and SNU-478:KDs was measured by the MTT assay. Cells plated in a 96-well plate were treated with gemcitabine (20 μg/mL), cisplatin (0.5 μg/mL), or 5-FU (50 μg/mL), and the MTT assay was carried out on day three of the treatments. (ANOVA P = 0.000 for gemcitabine; ANOVA P = 0.048 for cisplatin; ANOVA P = 0.001 for 5-FU).