Fig. 2

Targeting PAK1 by shRNA suppresses ESCC cell growth. a Western blotting analysis of PAK1 and p-PAK1 (T423) in single clones (PAK1#1 and PAK1#2) stably transfected with PAK1 in EC109 and KYSE70 cells. b Growth curve of indicated stable PAK1-overexpressing ESCC cells was detected by MTT cell proliferation assay (n = 8 per group). Representative images (left) and summary bar chart (ritht) of focus formation assay (n = 6 per group) (c) and colony formation in soft agar assay (n = 6 per group) (d) in ESCC cells stably transfected with PAK1 or empty vector (control). Scale bars, 200 μm. *P < 0.05, **P < 0.01 and ***P < 0.001 vs Vector group; P values were obtained by one-way ANOVA with post-hoc intergroup comparison with the Tukey’s test. e The effect of PAK1-targeting shRNAs was confirmed by Western blotting analysis. KYSE30 and KYSE150 were transfected with scrambled shRNA (shNC) or two shRNAs (shPAK1#1 and shPAK1#2) against PAK1. (f) The proliferation rate of the indicated stable PAK1-downregulated ESCC cells was examined by MTT assay (n = 8 per group). Silencing PAK1 could significantly decrease the frequency of focus formation (n = 6 per group) (g) and colony formation in soft agar (n = 6 per group) (h). Quantification (right panel) of colonies are depicted in the bar chart. Scale bars, 200 μm. *P < 0.05, **P < 0.01, ***P < 0.001 vs shNC; P values were obtained by one-way ANOVA with post-hoc intergroup comparison with the Tukey’s text