Figure 2.

BECN1 downregulation significantly impairs the advanced stages of CCCP-induced mitophagy. shSCR and shBECN1 SH-SY5Y cells stably expressing HA-PARK2 (A-D) or YFP-PARK2 (E and F) were treated with 25 µM CCCP up to 12 h. (A) Western blot of PARK2, LC3 and late mitophagic markers (TIMM23 and COX4I1). Separated lines indicate that we joined together distant parts from the same gel. (B and C) Densitometric analysis of data from (A). Values were normalized against TUBA/tubulin. (D) Analysis of mtDNA levels by RT-PCR. (E) Confocal analysis of shSCR and shBECN1 SH-SY5Y cells transfected with plasmids encoding YFP-PARK2 and DsRed-Mito after 12 h, 25 µM CCCP exposure. Scale bar: 10 µm. (F) Histogram reporting the statistical analysis of data from (E), expressed as mean ± SD of the percentage of cells with no mitochondria. *p<0.05; **p<0.001.