Fig. 2: Evaluation of mitophagy stimulation capacity of the AI top-scored molecules in vitro (mt-Keima) and in animals (mt-Rosella).
a, A schematic representation showing mechanisms of how the mt-Keima protein can be used as a mitophagy reporter. For confocal microscopy, dual-excitation ratio imaging was carried out with two sequential excitation lasers (458 nm and 561 nm). Representative confocal images are of HeLa cells expressing mt-Keima treated with vehicle (DMSO) or Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) (15 μM, 3 h). b,c, Effects of Quercetin, Tacrolimus, Ascomycin, Isorhamnetin, Pinostilbene, Kaem and Rhap (from 0.1 μM to 100 μM, 24 h) on mitophagy induction. d, Effects of in vitro-positive mitophagy inducers on the induction of neuronal mitophagy in worms expressing mt-Rosella reporter. Rotenone (5 μM and 10 μM, 4 h) was used as positive control. Data were pooled from 2 biological replicates (total n = 20–35 nematodes per group), with results shown as mean ± s.e.m. Two-way ANOVA followed by Tukey’s multiple comparisons test; NS, no significance; *P < 0.05, **P < 0.01, ***P < 0.001. A set of representative images of cellular positive (related to Fig. 2b,c) and negative mitophagy inducers (with quantifications) is included in Supplementary Fig. 2. Mechanisms of the mt-Rosella sensor as well as a set of representative images (related to Fig. 2d) are shown in Supplementary Fig. 4.
