Fig. 5

PROCA1 interacts with ZFP36L2 to decrease BCL2 mRNA stability and activate the mitochondrial apoptosis pathway. a. QRT-PCR (upper) and Western blot (lower) assays showing the expression level of ZFP36L2 in A549-DDP and PC9-DDP cells and their parental cell lines. Student’s two-tailed t-test, **P < 0.01. b. Co-IP experiments proved the interaction of endogenous ZFP36L2 and PROCA1 in A549/PC9 and A549-DDP/PC9-DDP cells. c. Co-localization of ZFP36L2 and PROCA1 in A549-DDP and PC9-DDP was detected by confocal immunofluorescence microscopy (scale bar, 10 μm). d. Schematic of ZFP36L2 binding to the 3' untranslated region of BCL2. e. RIP assay and agarose gel electrophoresis were used to corroborate the binding of ZFP36L2 protein to BCL2 mRNA. Student’s two-tailed t-test, **P < 0.01; ***P < 0.001. f. The interference efficiency of siRNA to ZFP36L2 was detected by western blot assays. GAPDH was used as a loading control. g. After ACT-D (1 μg/ml) treatment, BCL2 mRNA degradation was monitored by qRT-PCR in A549/PC9-siZFP36L2 or A549/PC9-control cells. Student’s two-tailed t-test, **P < 0.01; ***P < 0.001. h. Apoptosis-related proteins, including PARP, cleaved-PARP (c-PARP), Caspase3, cleaved-Caspase3 (c-CASP3) and BCL2 were analysed by western blot assays in ZFP36L2 inhibited A549 and PC9 cells. GAPDH and β-Tubulin were used as loading controls. Error bars, mean ± SD