Figure 6. KP46 activates autophagy, PARKIN and ubiquitination.

a–b. KP46 upregulates autophagic markers. a. Immunoblot analysis of LC3, ATG7 and BECLIN 1, BNIP3L. HCT116^WT cells exposed 6 hours to vehicle, or 10 μM KP46 or 200 nm Rapamycin. β-Tubulin served as loading control. b. Shown are the relative protein densities normalized to β-Tubulin. n = 3 (n = 5 for Bnip3L in the KP46 treated fraction), ±SEM, two-way ANOVA, Bonferroni's multiple comparisons test. ****p < 0.0001, ***p < 0.001, *p < 0.05 c. HCT116^WT cells were co-transfected with PARKIN-EYFP (shown in green) and mtRFP (red) and exposed to vehicle or 2.5 μM KP46 for 4 hours or 50 μM CCCP as indicated respectively, for 2 hours and immediately monitored under confocal microscopy. Yellow overlay indicates the co-localisation of green and red fluorescence. Scale bars: 10 μm. d–e. KP46 activates PARKIN. HCT116^WT cells were treated with vehicle, KP46 or CCCP and (d) immunoblotted for PARKIN and β-Tubulin and (e) quantified. n = 3, ±SEM, one-way ANOVA, Bonferroni's multiple comparisons test. **p < 0.01. f–g. KP46 triggers ubiquitination of proteins. Mitochondrial proteins were isolated from HCT116^WT cells. A gel was stained with Coomassie blue as loading control, and in parallel samples were (f) immunoblotted against Ubiquitin and COXIV, as mitochondrial marker, and (g) quantified. n = 3, ±SEM, one-way ANOVA followed by Bonferroni's multiple comparisons test. *p < 0.05.