Fig. 7: Autophagy signalling molecules involved in the climacostol regulation of autophagy.

a B16-F10 cells were cultured with 30 μg/ml climacostol (CLIMA) or control vehicle (CTRL) for increasing times. Western blotting images of phosphorylated Akt, S6 and AMPKα. The total Akt, S6 and AMPK were used as internal standard. Images are representative of 6 independent experiments. b Western blotting images of phosphorylated S6 and AMPK in subcutaneous B16-F10 melanoma allografts excised from mice at day 16 of treatment (from day 0 - every 3–4 days) with 100 µl CLIMA (600 μg/ml) or CTRL. The total S6 and AMPK were used as internal standard. Images represent the results obtained from 6 animals per experimental group. c Western blotting images of AMPKα, LC3 and p62 expression in B16-F10 cells transfected for 48 h with an AMPKα-specific (AMPKα siRNA) or a non-targeting siRNA (nt siRNA), followed by CLIMA treatment (24 h, 30 μg/ml). LDH was used as internal standard. Right panels: densitometric analysis of AMPKα, LC3-II and p62 relative to their respective standard. Results are expressed as fold change of nt siRNA + CLIMA. Images and data are representative of 3 independent experiments. *p < 0.05 and **p < 0.005 relative to nt siRNA + CLIMA. d Western blotting images of phosphorylated Akt, S6 and AMPKα in B16-F10 cell transfected for 48 h with a p53-specific (p53 siRNA) or a non-targeting siRNA (nt siRNA), followed by vehicle or CLIMA treatment (6 h, 30 μg/ml). The total Akt, S6 and AMPKα were used as internal standard. Right panels: densitometric analysis of phosphorylated proteins relative to their respective standard. Results are expressed as fold change of nt siRNA. Images and data are representative of 3 independent experiments. **p < 0.005 and ***p < 0.0001 relative to nt siRNA; #p < 0.05, ##p < 0.005 and ###p < 0.0001 relative to p53 siRNA, §p < 0.05 relative to nt siRNA + CLIMA. e Western blotting images of AMPKα and p53 expression in B16-F10 cells transfected for 48 h with an AMPKα-specific (AMPKα siRNA) or a non-targeting siRNA (nt siRNA), followed by vehicle or CLIMA treatment (24 h, 30 μg/ml). LDH was used as internal standard. Images are representative of 3 independent experiments