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. 2020 Nov 12;11:5731. doi: 10.1038/s41467-020-19547-6

Fig. 1. UMI-77 can induce mitophagy, without inducing mitochondrial damage or apoptosis.

Fig. 1

a 2024 FDA-approved drugs or drug candidates were used to screen for mitophagy inducers. Each color block represents a drug in the screen. Bcl-2 family proteins inhibitors are indicated in red. b HEK293T and HeLa cells treated with UMI-77 (5 µM) or CCCP (10 µM) for 12 h. Mitochondrial membrane potential was assessed using JC1. One-way ANOVA (data represent mean ± S.E.M.; HEK293T: NC (n = 24), UMI-77 (n = 45), CCCP (n = 12); HeLa: NC (n = 12), UMI-77 (n = 12), CCCP (n = 8). ****p < 0.0001. ns, not significant). c HEK293T-mt-Keima cells were treated with UMI-77 (5 µM) combined with CCCP (10 µM) for 4 or 6 h. The mitophagy levels were analyzed by one-way ANOVA (data represent mean ± S.E.M. The sample size was, in turn, n = 8, n = 6, n = 8, n = 5, n = 8, n = 7, n = 7. ****p < 0.0001, ***p < 0.001, *p < 0.05). d HEK293T cells were transfected with pcDNA3.1-mt-Keima (mitophagy reporter) plasmid and treated with UMI-77 at indicated concentrations with or without Z-VAD-fmk (50 µM) for 12 h. Cell viability was determined using LIVE/DEAD™ imaging kit. One-way ANOVA (data represent mean ± S.E.M.; n = 4. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. ns, not significant). e HEK293T-mt-Keima cells were stained with LysoTracker Green for 30 min, treated with 5 µM UMI-77. Scale bars, 5 µm. Quantification of the number of LysoTracker-positive dots colocalized with mt-Keima in cells were analyzed by two-tailed t test. (mean ± S.E.M.; DMSO (n = 10), UMI-77 (n = 14). **p < 0.01 (P = 0.0021)). f HeLa cells were treated with UMI-77 and Bafilomycin A1 (Baf-A1) for 8 h and analyzed by transmission electron microscopy. Scale bars, 5 µm; insets: Scale bar, 1 μm. g 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 (Tom20/Tubulin, Tim23/Tubulin). h cells were treated with 5 µM UMI-77 in the presence or absence of MG-132, E64D, and NH4Cl/Leupeptin (Leup) for 12 h, and the mitochondrial marker proteins (Tom20, Tim23) were detected by western blotting. The numbers under the blots represent the gray scale quantification (Tom20/Tubulin, Tim23/Tubulin). Source data are provided as a Source Data file.