Figure 1.

BECN1 is not required to trigger the early steps of mitophagy. (A and B) Confocal analysis of PINK1 accumulation and PARK2 recruitment at mitochondria upon CCCP treatment in shSCR and shBECN1 SH-SY5Y cells, overexpressing PINK1-GFP (A) or YFP-PARK2 (B). Cells were treated with DMSO alone or with 25 μM CCCP for 3 h and mitochondria were immunostained using the mitochondrial marker TOMM20 (red). Scale bar: 10 μm. (C and D) Statistical analysis of data from A-B (mean ± SD of n = 3, 30 cells per experiment). Histograms report the percentage of cells showing colocalization of PINK1 (C) or PARK2 (D) with mitochondria. **p < 0.001. (E) WB of PINK1 and early mitophagic markers in shSCR and shBECN1 SH-SY5Y cells overexpressing HA-PARK2. Degradation (shorter exposure) and ubiquitination (longer exposure, upper band) of the mitochondrial proteins MFN2, OPA1 and VDAC1 were assessed at the indicated early time points of treatment with 25 μM CCCP. Values were normalized against TUBA/tubulin. Separated blots indicate that we joined together distant parts from the same gel.