Fig. 7. Phosphorylation at Ser¹⁵ and localization of Beclin1 to MAMs during mitophagy.

A) Schematic illustration of the split GFP MAM reporter. B) Representative images of WT and Becn1^−/− MEFs overexpressing the MAM reporter and treated with FCCP (10 μM for 6 h). C) Quantification of the average number of MAMs/cell (a total of 30 cells were scored in 5 independent experiments). D) Representative images of WT and Becn1^−/− MEFs overexpressing the omegasome reporter mCherry-DFCP1. Cells were treated with FCCP (25 μM) for 4h and stained with anti-Calreticulin and anti-Tom20 to label ER and mitochondria, respectively. Quantification of total mCherry-DFCP1 punctate (white bars) and area colocalized with MAMs (grey bars) (n=90 cells/group from 3 independent experiments). E) Transmission electron microscopy images of WT and Becn1^−/− MEFs under baseline conditions and after treatment with 10 μm FCCP. Mitochondria are marked with M, red arrows mark small vesicular structures near mitochondria after FCCP treatment, and blue arrows mark larger vesicles. Scale bar = 500 nm. Data are presented as means ± SEM. **p<0.01, ***p<0.001, and ****p<0.0001 by two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Scale bars = 10 μm.