Fig. 1

RBM4 exhibits oncogenic activity in ESCC. a RBM4 protein levels were measured in 75 pairs of ESCC and normal esophageal tissues by immunohistochemistry staining of tissue microarray. Quantification of the percentage of cases exhibited negative, 1 + , 2+ or 3+ IHC staining of RBM4 was plotted. “−” indicates negative staining; “+” means weak positive staining; “++” denotes moderately strong positive staining, and “+++” indicates strong positive staining. Scale bar = 50 μm. b Immunohistochemistry and HE staining of normal esophageal tissues and pre-cancerous lesions of ESCC were shown. Scale bar = 10 μm. Quantitative analysis of RBM4 expression was done by Image J program. Plotted are the mean ± SD from five pairs of human samples, with *P < 0.05 as determined by paired Student t-test. H-score = ∑(pi × i) = (percentage of weak intensity × 1)+(percentage of moderate intensity × 2) + (percentage of strong intensity × 3), pi indicates the percentage of positive signal pixel area/number of positive tumor cells, i represents the coloring intensity. c Kaplan–Meier curve with corresponding 95% confidence intervals (CI) showing overall survival of ESCC patients with high or low RBM4 expression based on immunohistochemical microarray analysis. Dotted red/blue indicates the upper and lower confidence limits. Significance was assessed with Mantel-Cox log-rank (P = 0.0272) test. The hazard ratio for risk of death in RBM4-low versus RBM4- high ESCC patients is 0.5636 (95%CI = 0.375-0.9412). d Cell viabilities of RBM4-overexpressed KYSE150, KYSE30 and KYSE450 cells were measured by the CCK8 growth curve 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 < 0.001, ****P < 0.0001. e, f Nude mice were subcutaneously inoculated with KYSE150 or KYSE30 cells with stable knockdown of RBM4 or control respectively. Images of the subcutaneous xenograft tumors were shown in e. Tumor weight (e) and tumor volumes (f) of each group was measured and quantified at the indicated time. P-values were determined by Student’s t-test (e) or two-way repeated measures ANOVA (f). (n = 10, error bars indicate mean ± SEM, *P < 0.05, **P < 0.01.) g KEGG pathway analysis of RNA-seq with three independent biological replicates using KYSE150 cells with stable depletion of RBM4 compared to control vector. Statistical analysis was performed with the false discovery rate (FDR) method after Benjamini-Hochberg correction for multiple-testing. KEGG terms for biological process with FDR < 0.05 were accepted as a significant enrichment. h The morphology changes of KYSE30 and KYSE150 cells with stable knockdown of RBM4 by shRNA (upper panel) or knockout of RBM4 established by CRISPR-Cas9 system (lower panel). Scale bar = 25 μm. i β-gal staining of KYSE30 and KYSE150 cells with stable depletion of RBM4 by shRNA. Three experiments were carried out with mean ± SD of β-gal positive cells plotted. ****P < 0.0001 (P-values were determined by One-way ANOVA with Dunnett multiple comparisons). Scale bar = 25 μm. j β-gal staining of RBM4-knockout KYSE30 and KYSE150 cells generated through CRISPR-Cas9 system. Scale bar = 25 μm