Fig 1: CPT1C-induced EMT enhances cisplatin resistance. (A) Using the lung adenocarcinoma database (TCGA, Nature 2014, sample: 230) in the website (http://www.cbioportal.org/), the correlations between the expression of the CPT1C gene and the expression of SNAI1, CDH1, CDH2, and FN1 were analyzed. (B) After transfection of A549 and H1299 cells with CPT1C cDNA for 24 h, Western blot analysis was performed to detect CPT1C, Snail, fibronectin, and E-cadherin protein expression. (C) The cells were transfected with CPT1C siRNA for Western blot analysis of CPT1C, Snail, fibronectin, and E-cadherin expression. (D) After transfection of A549 and H1299 cells with CPT1C cDNA, wound healing assays were performed at various times (0, 24, 48 h) to determine the migration ability. The bars represent the mean ± S.D. of triplicates (**P < 0.01 for the difference from untreated control at 24 h; #P < 0.05 and ##P < 0.01 for the difference from untreated control at 48 h by ANOVA with Dunnett’s correction for multiple comparisons). (E) After transfection of A549 and H1299 cells with CPT1C siRNA, wound healing assays were performed. The bars represent the mean ± S.D. of triplicates (*P < 0.05 and **P < 0.01 for the difference from untreated control at 24 h; and ##P < 0.01 at 48 h by ANOVA with Dunnett’s correction for multiple comparisons). (F) A549 and H1299 cells were co-transfected with Snail promoter and control Renilla luciferase reporter gene plasmid and treated with the indicated cisplatin concentrations (0, 5, and 10 μM) after 48 h. Luciferase activity was determined and normalized using the dual luciferase reporter system. The bars represent the mean ± S.D. of triplicates (*P < 0.05 and **P < 0.01 for the difference from DDP-untreated A549 and H1299 cells by ANOVA with Dunnett’s correction for multiple comparisons). (G) The cells were untreated or treated with the indicated doses of cisplatin (0, 5, 10, 15, 20, and 25 μM) for 6 h. Then, Western blot analysis was performed to determine CPT1C, Snail, fibronectin, and E-cadherin. (H) After transfection of A549 and H1299 cells with CPT1C siRNA for 24 h, the cell lines were co-transfected with Snail promoter and control Renilla luciferase reporter gene plasmid and treated with the indicated cisplatin concentration (10 μM) for 48 h. Luciferase activity was determined and normalized using the dual luciferase reporter system. The bars represent the mean ± S.D. of triplicates (**P < 0.01 and ***P < 0.001 for the difference from untreated cells; and ###P < 0.001 for the difference from transfection with CPT1C siRNA cells by ANOVA with Dunnett’s correction for multiple comparisons). (I) After transfection of A549 and H1299 cells with CPT1C siRNA for 24 h, Western blot analysis was performed to determine CPT1C, Snail, fibronectin, and epithelial marker (E-cadherin) expression in A549 and H1299 cells treated with cisplatin (0, 5, and 10 μM) for 6 h. (J) After transfection of A549 and H1299 with CPT1C cDNA for 24 h, the cell lines were co-transfected with Snail promoter and control Renilla luciferase reporter gene plasmid and treated with 10 μM cisplatin for 48 h. Luciferase activity was determined and normalized using the dual luciferase reporter system. The bars represent the mean ± S.D. of triplicates (*P < 0.05 and ***P < 0.001 for the difference from untreated cells and #P < 0.05 and ##P < 0.01 for difference from transfection with CPT1C cDNA cells by ANOVA with Dunnett’s correction for multiple comparisons). (K) After transfection of A549 and H1299 cells with CPT1C cDNA for 24 h, Western blot analysis of CPT1C, Snail, fibronectin, and E-cadherin in A549 and H1299 cells treated with cisplatin (0, 5, and 10 μM) for 6 h. (L) Transfection of CPT1C siRNA alone or CPT1C siRNA combined with Snail cDNA, the A549 cells were treated with different concentrations of cisplatin for 48 h in preparation for MTT analysis. The bars represent the mean ± S.D. of triplicates (***P < 0.001 for the difference of CPT1C siRNA + Snail cDNA from the control group; and ###P < 0.001 for the difference in CPT1C siRNA from the control group by ANOVA with Dunnett’s correction for multiple comparisons).