Fig. 4: UMI-77 induces mitophagy in an ATG5-dependent manner, independent of adapter proteins NBR1, TAX1BP1, p62, and NDP52.

a PLA assay for MCL-1 and LC3A were performed in HeLa WT and quadruple KOs (NBR1, TAX1BP1, p62, and NDP52 knockout) cells treated with UMI-77 (5 µM) for the indicated times. Cells were analyzed by fluorescence microscopy. Scale bar, 20 μm. b Quantification of the mean area dots from a by two-tailed t test (data represents mean ± S.E.M. The sample size was, in turn, n = 49, n = 23, n = 60, n = 55 cells, ***p < 0.001). c HeLa WT and quadruple KOs (NBR1, TAX1BP1, p62, and NDP52 knockout) cells were treated with 5 µM UMI-77 for the indicated times and cell lysates were immunoblotted with indicated antibodies. The numbers under the blots represent the gray scale quantification (Cox II/Tubulin, Tim23/Tubulin). d MEF WT and ATG5 knockout cells were treated with 5 µM UMI-77 for the indicated times, and mitochondrial marker protein Tim23 and LC3 were detected by western blotting. The numbers under the blots represent the gray scale quantification (Tim23/Tubulin). e The mitophagy levels of control cells and ATG5 knockdown cells treated with UMI-77 were analyzed using one-way ANOVA (data represent mean ± S.E.M.; n = 6. ****p < 0.0001. ns, not significant). The siRNA knockdown efficiency was shown using western blot. siNC: scrambled siRNA. Source data are provided as a Source Data file.