Fig. 5: Intricacies of mitophagy regulation and its ramifications on cellular demise.

a Schematic diagram of HCQ blocking mitophagic flux. b CLSM images of fluorescent co-localization of mitochondria (Mito-Tracker Red) and autophagosomes (Green-LC3II). PCC, Pearson Correlation Coefficient. Scale bar = 40 μm. TEM images of c B16 and d MCF-7 cells after different treatments under hypoxia (Yellow arrow: autophagosomes; Red arrow: autolysosomes). Scale bar = 4 μm or 1 μm. Immunofluorescence images of the key protein change in e B16 and f MCF-7 cells for mitophagy, including PINK1 and Parkin, after different treatments under hypoxia. PBS-N represents the PBS group cultured under normoxic conditions (Blue: DAPI; Green: Mito-Tracker; Orange: PINK1; Red: Parkin). Scale bar = 20 or 4 μm. Three independent experiments in b–f were performed with similar results. g Intracellular ATP levels in B16 and MCF-7 cells following treatment with PBS, HCQ, HC4H, SHC4+hv, and SHC4H+hv (HCQ: 10 μM) treatments (n = 3 independent experiments; mean values  ±  SD). Statistical differences were determined by One-way ANOVA with a Tukey post-hoc test. h The expression of key proteins for mitophagy in B16 and MCF-7 cells, including LC3II/I, p62, after different treatments. NAC, N-acetylcysteine (5 mM). At least four independent experiments were performed and representative results are shown. After different treatments, apoptosis analysis by flow cytometry in i B16, j MCF-7 cells. Three independent experiments were performed and representative results are shown. Source data are provided as a Source Data file.