Extended Data Fig. 1: CISD1-RFP-SQSTM1 induces forced mitophagy and reduces the mitochondrial number.
From: Reduction of mtDNA heteroplasmy in mitochondrial replacement therapy by inducing forced mitophagy
a. HeLa cells were stably transfected with GFP-LC3 (green) and stained with MitoTracker Deep Red FM (grey) to label mitochondria. CISD1-RFP-SQSTM1 (red) was transiently transfected into the HeLa cells. Images were obtained 60 hours after CISD1-RFP-SQSTM1 transfection. The insets indicate that CISD1-RFP-SQSTM1-labeled mitochondria co-localizes with GFP-LC3. b. CISD1-RFP-SQSTM1-labelled mitochondria fuse with lysosomes. HeLa cells were stably transfected with pmEmerald-Mito (grey) to label mitochondria, transiently transfected with CISD1-RFP-SQSTM1 (red), and stained with LysoTracker Blue DND-22 (green) to label lysosomes. Images were obtained 48 hours after CISD1-RFP-SQSTM1 transfection. The insets indicate that CISD1-RFP-SQSTM1-labeled mitochondria fuse with lysosomes. c. The CISD1-RFP-SQSTM1-expressed cells have fewer mitochondria than the unexpressed cells. The stably pmEmerald-Mito (green)-transfected HeLa cells were transiently transfected with CISD1-RFP-SQSTM1 (red) and stained with Hoechst 33342. Images were obtained24 and 48 hours after CISD1-RFP-SQSTM1 transfection. White arrowheads indicate that the CISD1-RFP-SQSTM1-expression cells decrease the pmEmerald-Mito fluorescence signals. In a–c, scale bars, 30 μm, and three independently repeated experiments with similar results. d. The fluorescence analysis of mitochondria in the CISD1-RFP-SQSTM1-expressed and control cells at different time points. Data were analyzed by two-tailed unpaired t-test and expressed as average ± SEM, and each point represents one cell from 3 biological replicates. For the 24 hours group, n = 106, the control value is 945 ± 84.97, and the CISD1-RFP-SQSTM1 value is 950.8 ± 90.19. For the 48 hours group, n = 67, the control value is 815.9 ± 51.32, and the CISD1-RFP-SQSTM1 value is 455.7 ± 39.86. e. The mtDNA number decreased after CISD1-RFP-SQSTM1 expression. Forty-eight hours after CISD1-RFP-SQSTM1 transfection, a single cell with or without CISD1-RFP-SQSTM1 expression was picked up, and the mtDNA quantity was analyzed by qPCR. Data were analyzed by two-tailed unpaired t-test and expressed as average ± SEM. n = 27 for control, and n = 30 for CISD1-RFP-Binding from 3 biological replicates. The value of control is 2357 ± 104.3, and the value of CISD1-RFP-Binding is 1274 ± 79.14.
