Fig. 7

The microprotein TPM3P9 inhibits RBM4-mediated TCF7L2 RNA splicing. a Western blot results showing that overexpression of RBM4 dramatically abrogated the TPM3P9-mediated increase in TCF7L2-L protein expression. b qRT-PCR results showing that RBM4 dramatically abrogated the TPM3P9-mediated increase in TCF7L2-L mRNA expression. ***p < 0.001. c RT-PCR results showing that the increase in the TCF7L2-L/TCF7L2-S ratio induced by TPM3P9 overexpression was reversed to the control level when RBM4 was overexpressed. **p < 0.01; ***p < 0.001; ns, nonsignificant. d Immunofluorescence staining showing that TPM3P9 overexpression did not affect the cellular localization or expression of the RBM4 protein. Nuclei were stained with DAPI (blue), TPM3P9 (green), and RBM4 (red). The staining intensity of RBM4 (red) was quantified. Unpaired two-tailed Student’s t-test; ns, nonsignificant. Bars, SEMs; scale bars, 10 μm (left) and 20 μm (right). e Western blot results showing that TPM3P9 overexpression did not affect the expression of the RBM4 protein in ccRCC cells. f RIP assay results showing that TPM3P9 overexpression weakened the binding ability of RBM4 to TCF7L2 pre-mRNA in ccRCC cells. g Overexpression of RBM4 restored the TPM3P9-mediated promotion of ccRCC cell growth. ***p < 0.001. h Mouse xenograft model showing that overexpression of RBM4 significantly inhibited tumor growth promoted by TPM3P9 overexpression. **p < 0.01; ns, nonsignificant. i, j Mouse xenograft model established with ACHN cells. Tumor growth (i), and tumor weight (j) are shown. Data are shown as the mean ± SD for 5 mice per group. Unpaired two-tailed Student’s t-test; **p < 0.01. k The body weight of the nude mice was monitored. ns, nonsignificant. l Co-IP assay results verifying the interaction between RBM4 and TPM3P9 in in vivo tumor samples. m Western blot results showing that RBM4 overexpression abolished the upregulation of TCF7L2-L mediated by TPM3P9 in in vivo tumor samples. n Kaplan–Meier survival analysis results showing that high RBM4 expression was associated with favorable overall survival in ccRCC patients in the SYSUCC cohort (p < 0.05, log-rank test). o Kaplan–Meier survival analysis showing that high RBM4 expression was associated with favorable disease-free survival in ccRCC patients in the SYSUCC cohort (p < 0.05, log-rank test). p Immunohistochemical data revealing the correlation between TPM3P9 and RBM4 expression in ccRCC patients. Scale bars, 50 μm. q Kaplan–Meier analysis of overall survival revealed that in the SYSUCC cohort, ccRCC patients with both high TPM3P9 and low RBM4 expression had the worst prognosis, while patients with both low TPM3P9 and high RBM4 expression had the best prognosis (p < 0.05, log-rank test). r Kaplan–Meier analysis of disease-free survival showed that in the SYSUCC cohort, ccRCC patients with both high TPM3P9 and low RBM4 expression had the worst prognosis, while patients with both low TPM3P9 and high RBM4 expression had the best prognosis (p < 0.05, log-rank test)