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. 2022 Apr 13;18(12):2985–3003. doi: 10.1080/15548627.2022.2062111

Figure 8.

Figure 8.

The LC3A-EE double mutation that hampers LC3A binding to CL in vitro also decreases its location to mitochondria in rotenone- and CCCP-induced mitophagy. SH-SY5Y cells were co-transfected with DsRed2-Mito7 (DsRed) and GFP-tagged WT or mutant LC3A. Vehicle (Veh) controls were treated with DMSO. (A) Percent GFP-LC3A or GFP-LC3A-EE puncta that colocalize with mitochondria, a signal of mitophagy, in SH-SY5Y cells untreated (vehicle), or treated with rotenone (1 μM, 6 h), or CCCP (20 μM, 1 h) or O/A (10/4 μM, 6 h). To compute the percent colocalization, images were analyzed with the JACop plugging of ImageJ. At least 30 images were analyzed per condition. *P < 0.05, ns: non-significant. (B) Representative images of GFP-LC3A and GFP-LC3A-EE SH-SY5Y transfected cells treated with 1 μM rotenone for 6 h. (C) Representative images of GFP-LC3A and GFP-LC3A-EE SH-SY5Y transfected cells treated with 20 μM CCCP for 1 h. (D) Representative images of GFP-LC3A and GFP-LC3A-EE SH-SY5Y transfected cells treated with O/A (10/4 μM for 6 h). Images were acquired using a Nikon Eclipse C1 confocal microscope. Scale bar: 10 μm. At the right-hand side of each condition, DsRed (red) and GFP (green) line profiles show examples of co-localization and non-colocalization events.