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. 2012 Mar 1;8(3):e1002537. doi: 10.1371/journal.pgen.1002537

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Liu et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Time course of CCCP treatment to monitor mitophagy in Hela cells stably transfected with Venus-Parkin. Cells were transfected with control siRNA or hMiro1+hMiro2 siRNAs and treated with 10 µM CCCP for the indicated time. Markers for mitochondrial outer membrane (Tom20, VDAC1), intermembrane space (HtrA2/Omi, Cyt c), inner membrane (Tim23, NDUFA9), and matrix (PDHA1, Hsp60) were examined by Western blot to determine the elimination of damaged mitochondria. β-actin was used as a loading control. (B) Immunofluorescence staining showing the formation of perinuclear mitochondrial aggregates after siRNA-mediated knockdown of hMiro1 or hMiro2. HeLa cells transfected with control siRNA, hMiro1 siRNA, hMiro2 siRNA, or hMiro1+hMiro2 siRNAs were stained for Tom20 and α-tubulin. Merged images are shown to the right. Insets show enlarged view of mitochondrial morphology in the boxed areas. Arrowheads indicate ring-like or round-shaped mitochondria. (C) Miro knockdown facilitates the early stage of mitophagy in venus-Parkin transfected HeLa cells treated with CCCP. HeLa cells stably transfected with venus-Parkin were co-transfected with control siRNA or hMiro1+hMiro2 siRNAs and then treated with the DMSO vehicle or CCCP for 90 mins or 3 hrs. Venus-Parkin (blue), Tom20 (green), and Hsp60 (red) were visualized by immunostaining. Merged Tom20/Hsp60 signals are shown to the right. Cells of interest are outlined.