Fig. 2

RBM4 depletion induces senescence-like phenotype and its overexpression contributes to bypass premature senescence. a β-gal staining of KYSE450 and KYSE510 cells with stable depletion of RBM4. 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. b The expression of senescence-associated secretory phenotype (SASP) factors in KYS150 and KYSE450 cells with stable depletion of RBM4 was examined with the qRT-PCR approach. P-values were determined by One-way ANOVA with Dunnett multiple comparisons. n = 3 per group. c ESCC cells expressing GFP were co-cultured with RBM4-depleted ESCC cells, and morphology changes of ESCC-GFP cells were observed. Scale bar = 50 μm. Mean ± SD of percentage of relative senescence cells were plotted. P-values were determined by One-way ANOVA with Dunnett multiple comparisons. n = 3 per group. d The relative ROS production was examined in KYSE150 and KYSE450 cells with depletion of RBM4. Three experiments were carried out with mean ± SD of relative ROS production was plotted. (P-values were determined by One-way ANOVA with Dunnett multiple comparisons). e, f β-gal staining of KYSE30, KYSE150 and NE2 cells with stable lentiviral transfection of RBM4 or empty vector with or without H-RAS overexpression (e) or Doxorubicin treatment (f). 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. g The protein levels of RBM4, p-Rb, cyclin D3, cyclin D1, CDK6, CDK4, P53, P21, and P27 in ESCC cells with RBM4 depletion were examined with a western blot assay. h β-gal staining of different cancer cells with distinct genetic background in the presence or absence of RBM4. Three experiments were performed with mean ± SD of β-gal positive cells plotted (P-values were determined by Student’s t-test) and the status of P53, INK4A, and K-Ras was indicated. Scale bar = 25 μm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001