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. 2022 Oct 18;23(12):e55191. doi: 10.15252/embr.202255191

Figure EV4. The effect of optineurin (OPTN) or NDP52 single knockdown on mitochondrial DAG production and double knockdown on mito‐autophagosome (mitophagosome) formation.

Figure EV4

  1. Hela cells were transfected with control siRNA, OPTN‐siRNA, or NDP52‐siRNA. Knockdown cells were subsequently transfected with the DAGR‐YFP reporter and FLAG‐Parkin, followed by CCCP treatment. Note that DAGR accumulated on mitochondria (Tom20) in OPTN and NDP52 single knockdown cells. Scale bar = 25 μM.
  2. OPTN and NDP52 double knockdown cells were transfected with GFP‐Parkin and RFP‐DAGR, followed by CCCP treatment (10 μM) for 9 h. Autophagosomes were assessed by immunostaining with an LC3 antibody. Note that DAG‐positive LC3 vesicle production was reduced in OPTN and NDP52 double knockdown cells. Scale bar = 25 μM, and zoom is 5×.
  3. Knockdown efficiency for OPTN and NDP52 was confirmed by immunoblotting.
  4. Hela cells were transfected with OPTN‐siRNA and NDP52‐siRNA followed by plasmids for FLAG‐Parkin, DAGR‐YFP, and OPTN‐WT or OPTN‐E478G mutant and treated with CCCP (5.5 h), as indicated. Note that OPTN‐WT, but not the ubiquitin‐binding deficient E478G mutant, restored mitochondrial DAGR‐YFP accumulation induced by CCCP. Scale bar: 10 mm.