Fig. 6

CPEB4 enhances cisplatin resistance by upregulating the expression of the antiapoptotic protein Mcl-1. A Western blot analysis of Mcl-1 in ESCC cells with CPEB4 overexpression or knockdown. B Relative mRNA level of Mcl-1 in ESCC cells with CPEB4 overexpression or knockdown. C A schematic diagram illustrating the putative CPEB4 binding motif in the 3’UTR of Mcl-1. D RIP assays were performed to detect the interaction between CPEB4 and Mcl-1 mRNA. Bottom, IP efficiency of the CPEB4 antibody. Top, the abundance of CPEB4-associated Mcl-1 mRNA was analyzed by qPCR and normalized to the input control. An IgG antibody was used as a negative control. E A biotinylated probe pulldown assay was performed to detect the interaction between CPEB4 and Mcl-1 mRNA. F Western blot analysis of Mcl-1 and caspase-3 expression in ECA109-CR cells in the presence or absence of the polyadenylation inhibitor cordycepin (5, 10 and 20 µg/mL). G FISH and IF images of CPEB4 and Mcl-1 mRNA in ESCC samples from cohort II. Scale bars, 100 μm. H ESCC cells in which CPEB4 was overexpressed or silenced were sequentially lysed, added to a sucrose gradient, and subjected to ultracentrifugation. The Mcl-1 mRNA levels in each fraction were determined by qPCR. I The viability of the indicated cells after treatment with cisplatin (10 µg/mL) was determined by the CCK-8 method. J Clonogenic assay of the indicated cells treated with increasing doses of cisplatin. K The apoptotic rates of the indicated cells after treatment with cisplatin (10 µg/mL) for 24 h as determined by Annexin V-FITC/PI FACS analysis. L The viability of the indicated cells after treatment with cisplatin (10 µg/mL) as determined by the CCK-8 method. M Clonogenic assay of the indicated cells treated with increasing doses of cisplatin. N The apoptotic rate of the indicated cells after treatment with cisplatin (10 µg/mL) for 24 h was determined by Annexin V-FITC/PI FACS analysis. O Correlation between cDOPEY2 RNA expression and CPEB4 protein levels (upper panel) and correlation between Mcl-1 protein abundance and CPEB4 protein levels (bottom panel) in cohort I. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined using the unpaired Student’s t-test (B) and one-way ANOVA with Tukey’s post hoc test (I, J, K, L, M and N). Correlations were determined by Spearman correlation analysis (O)