Fig. 3

AIL directly interacted with c-Jun and promoted its degradation. A SK-MEL-5 and B16-F10 cell lysates were detected by western blot with anti-c-Jun antibody for the pull-down assay. B Differential expression levels of c-Jun in PIG1, SK-MEL-5, SK-MEL-28, JB6, B16-F10 and YUMM1.7 cells. The c-Jun protein level in PIG1, SK-MEL-5, SK-MEL-28, JB6, B16-F10 and YUMM1.7 cells was measured by western blot. GAPDH was used as the loading control. C-D PIG1 and JB6 cells were transfected with c-Jun overexpression or negative control vector, and c-Jun expression was analyzed by western blot. GAPDH served as the loading control. Cells were then treated with various concentrations of AIL for 72 h, and cell viability was examined by CCK-8 assay. E SK-MEL-5 and B16-F10 cells were treated with the indicated concentrations of AIL for 24 h. QRT-PCR was performed to analyze c-Jun mRNA levels. F SK-MEL-5 and B16-F10 cells were treated with the indicated concentrations of AIL for 24 h. Western blot was applied to detect c-Jun expression. GAPDH was used as a loading control. G SK-MEL-5 and B16-F10 cells were treated with AIL for different lengths of time. Western blot was applied to detect the c-Jun protein level. GAPDH was used as a loading control. H Western blot analysis of the expression of c-Jun in tumor samples with the indicated concentrations of AIL treatment. GAPDH was used as a loading control. I SK-MEL-5 and B16-F10 cells were treated with cycloheximide (CHX, 200 µg/mL) with or without AIL for the indicated time. The c-Jun protein level was measured by western blot. GAPDH was used as a loading control. J SK-MEL-5 and B16-F10 cells were treated with AIL with or without MG-132 (30 µmol/L), and c-Jun protein levels were measured by western blot. GAPDH was used as a loading control. Data are shown as the mean ± SD, n = 3; ns, not significant; *p < 0.05, **p < 0.01 and ***p < 0.001