Fig. 3

RBM4 reduction activates LKB1-AMPK-P27 pathway to induce cellular senescence and growth arrest. a Protein levels RBM4, CDK4, CDK6, and P27 were analyzed in RBM4-depleted ESCC cells with or without P27. b β-gal staining of KYSE150 and KYSE450 cells with stable knockdown of RBM4 in the presence or absence of P27. Three experiments were carried out with mean ± SD of β-gal positive cells plotted (P-values were determined by One-way ANOVA with Dunnett multiple comparisons). Scale bar = 25 μm. c The growth curve of RBM4-depleted KYSE150 or KYSE450 cells with or without depletion of P27 was measured by CCK8 assay. P-values were determined by two-way repeated measures ANOVA. n = 3 per group. d Colony formation assays of RBM4-depleted KYSE150 or KYSE450 cells with or without knockdown of P27 were performed. The mean ± SD of colony numbers was plotted (P-values were determined by One-way ANOVA with Dunnett multiple comparisons, n = 3 per group). e Protein levels of P27, SKP2, p-S6K, p-4EBP1, p-AMPK, and RBM4 were examined in RBM4-depleted KYSE150 and KYSE450 cells with a western blot assay. f Western blot was performed to measure the protein levels of LKB1, p-AMPK, AMPK, and RBM4 in KYSE150 and KYSE450 cells with or without RBM4. g Protein levels of LKB1, p-AMPK, AMPK, p-S6K, p-4EBP1, and RBM4 were examined in RBM4-overexpressed KYSE150 and KYSE450 cells using a western blot assay. h Western blot approach was performed to measure the protein levels of LKB1, RBM4, and P27 in RBM4-depleted ESCC cells with or without LKB1. i Protein levels of p-Rb, P27, SKP2, p-AMPK, AMPK, and RBM4 were analyzed in RBM4-depleted ESCC cells with or without AMPK. j β-gal staining of KYSE150 cells with stable knockdown of RBM4 in the presence or absence of AMPK or LKB1. Three experiments were carried out with mean ± SD of β-gal positive cells plotted (P-values were determined by One-way ANOVA with Dunnett multiple comparisons). Scale bar = 25 μm. k, l Colony formation assays of RBM4-depleted KYSE150 cells with or without knockdown of AMPK or LKB1 were performed. Three experiments were carried out with mean ± SD of colony numbers were plotted. P-values were determined by One-way ANOVA with Dunnett multiple comparisons. m Cell viability of RBM4-depleted KYSE150 cells with or without AMPK or LKB1 was measured by CCK8 assays. Three experiments were carried out with mean ± SD of relative cell viability plotted. P-values were determined by two-way repeated measures ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001