Fig. 4

Raf1/MEK/ERK cascade is crucial for the aggressive cancer phenotype of PAK1 in ESCC cells. a Western blotting analysis was used to compare the protein levels of PAK1, Raf1, p-Raf1 (S338), MEK1, p-MEK1 (S298), ERK1/2, p-ERK1/2 (T202/Y204) in PAK1-overexpressing EC109 and KYSE70 cells, and in shPAK1-transfected KYSE30 and KYSE150 cells. Actin was used as loading control. b The effect of Raf1 shRNAs was confirmed by Western blot analysis (left). Silencing Raf1 significantly decreased the frequency of focus formation (middle) and colony formation in soft agar (right). Column and error bar represents mean ± SD (n = 6 per group). *P < 0.05, **P < 0.01, ***P < 0.001 by Student’s t test. c Raf1 knockdown dramatically reduced the migratory ability of PAK1-overexpressing EC109 and KYSE70 cells (left). Downregulation of Raf1 reduced the migratory (middle) and invasive abilities (right) of PAK1-overexpressing cells. Column and error bar represents mean ± SD (n = 6 per group). **P < 0.01, ***P < 0.001 by Student’s t test. d Knocking down of Raf1 attenuated MMP-2 and MMP-9 expression in PAK1-overexpressing EC109 and KYSE70 cells. e The efficiency of Raf1 overexpression in shPAK1-depleted KYSE30 and KYSE150 cells was confirmed by Western blot analysis (left). Enforced expression of Raf1 promoted the frequency of focus formation (middle) and colony formation in soft agar (right). Column and error bar represents mean ± SD (n = 6 per group). **P < 0.01, ***P < 0.001, one-way ANOVA with post-hoc intergroup comparison with the Tukey’s test. f Raf1 overexpression increased the migratory ability of shPAK1-depleted KYSE30 and KYSE150 cells (left). Raf1 overexpression enhanced the migratory (middle) and invasive abilities (right) of shPAK1-depleted KYSE30 and KYSE150 cells. Column and error bar represents mean ± SD (n = 6 per group). **P < 0.01, ***P < 0.001, one-way ANOVA with post-hoc intergroup comparison with the Tukey’s test. g Ectopic expression of Raf1 upregulated the protein levels of MMP-2 and MMP-9 in shPAK1-depleted KYSE30 and KYSE150 cells