Fig 2: JNK/c-jun pathway is activated simultaneously by Tivantinib.a The role of JNK/c-jun pathway in cell apoptosis. b JNK and its downstream proteins, p-JNK, JNK1, p-c jun, bim, and p-bad were analyzed using immunoblotting. CC cell lines were treated with Tivantinib 0.5 and 5 µM for 24, 48, and 72 h. c In Tivantinib (0.5 and 5 µM) short time exposure, the changes of p-JNK, JNK1, and p-c jun were evaluated using immunoblotting. The exposure times were 3, 6, 12, and 24 h. d–f Fluorescence microscopy features the localization of p-c jun/p-JNK and c-jun/JNK1 in HuCC-T1 cell. Magnification: × 20 and × 40 (insets)

Fig 3: 5-FU induces JNK activation and Bcl-2 phosphorylation in HCT116 p53-/- and HT-29 cells.(A) HCT116 p53-/- and HT29 cells were treated with varying concentrations of 5-FU for 24 h. The expression of several key MAPK regulators was examined by western blot (the blots were cropped, and the full-length blots are included in the supplementary information). (B) HCT116 p53-/- and HT29 cells were treated by 5-FU for 24 h, and then JNK, c-Jun and Bcl-2 phosphorylation, as well as the expression of active caspase-3 and several key autophagic regulators, were analyzed by western blotting.

Fig 4: JNK inhibition augments the cytotoxic effect of 5-FU in HCT116 p53-/- and HT29 cells by reducing survival autophagy.(A) HCT116 p53-/- and HT29 cells were treated with 5-FU with or without SP600125 for 24 h. Cells were fluorescently labeled and imaged using a confocal microscope. Green, FITC-labeled LC3; Blue, DAPI-labeled nucleus; quantitative analyses of punctate fluorescence numbers are shown; *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate. (B) JNK, c-Jun and Bcl-2 phosphorylation, as well as the expression of active caspase-3 and several key autophagic regulators, were analyzed in HCT116 p53-/- and HT29 cells treated with the combination of 5-FU and SP600125 (the blots were cropped, and the full-length blots are included in the supplementary information). (C) Cell viability was quantified by the MTT assay in these resistant cells treated with the combination of 5-FU and SP600125; *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate. (D) Clonogenic survival assay; *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate. (E) The expression of JNK in HCT116 p53-/- and HT29 cells was analyzed by RT-PCR when these cells were transiently transfected with the negative control siRNA siJNK (F) JNK, c-Jun and Bcl-2 phosphorylation, as well as the expression of active caspase-3 and several key autophagic regulators, were analyzed in HCT116 p53-/- and HT29 cells transiently transfected with the negative control siRNA siJNK (the blots were cropped, and the full-length blots are included in the supplementary information). (G) Cell viability was quantified by the MTT assay in these resistant cells transiently transfected with the negative control siRNA siJNK; *p < 0.05, compared with the 5-FU group. The experiments were performed in triplicate.

Fig 5: The synergistic activation of JNK/c-jun pathway plays a functional role in determining the apoptotic effect of Tivantinib.a Effect on the expression of JNK and c-jun by co-transfection of JNK-specific siRNA oligonucleotide sequences (each at the concentration of 25 and 50 nM) in HuCC-T1 and EGI-1 cell were assessed by western blotting; medium or non-coding siRNA (Ctrl-siRNA) were used as control. b Cell viability after transfection of siRNA targeting JNK in HuCC-T1 and EGI-1 cell. *p < 0.05 in comparison with cells treated by Tivantinib 0.5 µM for 24 h. FACS analysis of apoptosis ****p, ***p, **p < 0.01, and *p < 0.05 in comparison with cells with medium or with non-coding siRNA transfection. For this experiment, Tivantinib 0.5 µM was added into cell culture for 3 h after 24 h JNK siRNA transfection. c Cell viability analysis of HGF (25 and 50 ng/mL) stimulated cells treated by Tivantinib. p-MET, MET, p-JNK, and JNK1 were evaluated by western blotting in the same condition. HFG stimulated HuCC-T1 and EGI-1 cell were incubated with HGF for 3 h before being added to Tivantinib 0.5 and 5 µM for 24, 48, and 72 h. d Effect of c-jun silencing by specific siRNA in CC cells. e FACS analysis of apoptosis after transfection of siRNA targeting c-jun.****p, ***p, **p < 0.01, and *p < 0.05 in comparison with cells with medium or with non-coding siRNA transfection. f Western blotting analysis of p-MET, MET, p-JNK, JNK1, p-c jun FADD, and cleaved caspase-3 express in HuCC-T1 and EGI-1 cell treated by c-MET neutralizing antibody for 24, 48, and 72 h