Fig 2: 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 ß-Tubulin were used as loading controls. Error bars, mean ± SD

Fig 3: Low expression of LINC00173 in DDP-resistant LUAD attributes to the negative regulation of c-Myc. a. Predicted c-Myc binding site in the promoter region of LINC00173. b. Transfection of c-Myc plasmids suppressed LINC00173 expression in A549 and PC9 cells. Student’s two-tailed t-test, **P < 0.01; ***P < 0.001; ****P < 0.0001. c. ChIP assay and agarose gel electrophoresis verified the binding of c-Myc to LINC00173 promoter in A549 and A549-DDP cells. Student’s two-tailed t-test, **P < 0.01; ****P < 0.0001. d. Luciferase reporter assay further validated c-Myc was directly binding to LINC00173 promoter. Student’s two-tailed t-test, *P < 0.05; **P < 0.01; ns, no significance. e. Western blot analysis of PI3K, p-PI3K, AKT, p-AKT and c-Myc expression in A549 and A549-DDP cells. GAPDH was used as a loading control. f. Western blot detection of p-PI3K, p-AKT, c-Myc, PROCA1 and ZFP36L2 expression in A549-DDP and PC9-DDP cells after treated with PI3K inhibitor LY294002. GAPDH was used as a loading control. g. The expression level of LINC00173 was obviously increased in A549-DDP and PC9-DDP cell lines after treated with LY294002. h. The IC50 values to cisplatin were detected in A549 and PC9 cells transfected with a gradient concentration of c-Myc plasmids. Student’s two-tailed t-test, **P < 0.01; ****P < 0.0001. Error bars, mean ± SD

Fig 4: LINC00173 mediated chemosensitivity to DDP in LUAD via PROCA1/ZFP36L2/BCL2 axis. a. The protein level of PROCA1, BCL-2, PARP, cleaved-PARP (c-PARP), Caspase3 and cleaved-Caspase3 (c-CASP3) in LINC00173 transfected A549-DDP and PC9-DDP cells (cultured with cisplatin) were examined by western blot. GAPDH and ß-Tubulin were used as loading controls. b. Co-immunoprecipitation analysed the level of PROCA1 binding to ZFP36L2 in A549 and A549-DDP cells (cultured with cisplatin) transfected with shLINC00173 or LINC00173, respectively. c and d. BCL2 mRNA degradation was monitored by qRT-PCR in A549-shLINC00173 (c) or A549-DDP-LINC00173 cells cultured with cisplatin (d). e. The expression of LINC00173 in xenograft tumor tissues generated from A549 cells expressing either shRNAs targeting LINC00173 (shLINC00173-1) or nontargeting shRNA (scramble) and treated with cisplatin or control normal saline (N.S) f. The protein levels of PROCA1, ZFP36L2, and BCL2 in xenograft tumor tissues generated from A549 cells expressing either shRNAs targeting LINC00173 (shLINC00173-1) or nontargeting shRNA (scramble) and treated with cisplatin or control normal saline (N.S). Student’s two-tailed t-test, *P < 0.05; **P < 0.01; ***P < 0.001. Error bars, mean ± SD