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. Author manuscript; available in PMC: 2023 Mar 16.
Published in final edited form as: Sci Signal. 2023 Jan 31;16(770):eabo4457. doi: 10.1126/scisignal.abo4457

Fig. 4. Beclin1-deficient cells have reduced mitophagy.

Fig. 4.

A) Schematic illustration of the mitophagy reporter which is composed of a mitochondrial targeting sequence (MTS), mCherry (red), pHluorin2 (green) and a transmembrane domain (TM). B) Representative images of WT, BECN1−/− and BECN2−/− HeLa cells overexpressing the mitophagy reporter before and after FCCP (10 μM, 6h) treatment. C) Quantification of the number of mitophagy events per cell after FCCP treatment (n=90 cells/group from 3 experiments). D) Representative Western blots for COX IV in WT, BECN1−/− and BECN2−/− HeLa cells at baseline and after FCCP (25 μM) treatment (n=5 independent experiments). E) Quantification of COX IV protein levels in WT, BECN1−/− and BECN2−/− HeLa cells (n=5 independent experiments). F) Representative images and quantification of the number of mitophagy events per cell in WT and Becn1−/− MEFs overexpressing the mitophagy reporter (n=90 cells/group from 4 experiments). Cells were treated with FCCP (10 μM) for 6 h. G) Representative Western blots for COX IV and Tim23 in WT and Becn1−/− MEFs treated with FCCP (25 μM) for 0, 12 and 24 h. H) Quantification of COX IV and Tim23 protein levels in WT and Becn1−/− MEFs after 12 h of FCCP treatment (n=5 independent experiments). Data are presented as means ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Scale bars = 10 μm.