Figure 2. The BH3 domain of BCL2L1 is critical for mitophagy inhibition. (A) HeLa cells stably expressing RFP-LC3 were cotransfected with MYC-tagged FUNDC1 and GFP-tagged BCL2L1 or BCL2L1 deletion mutants. At 24 h post-transfection, the fixed cells were immunostained with an anti-MYC antibody (blue). Representative confocal microscopy images are presented. Scale bar: 10 μm. (B) Experiments were performed as described in (A), and the percentage of MYC-tagged FUNDC1-positive cells with LC3 puncta was quantified. Data represent mean ± s.e.m of 3 independent experiments (n > 100 cells per condition in each experiment). (C) HeLa cells were cotransfected with MYC-tagged FUNDC1 and GFP-tagged BCL2L1 or BCL2L1 mutants. At 24 h post-transfection, the cell lysates were analyzed with the indicated antibody. Grayscale values of the TOMM20 and TIMM23 bands measured with ImageJ are shown under the corresponding bands to indicate the band intensities. (D) HeLa cells stably expressing RFP-LC3 were cotransfected with MYC-tagged FUNDC1 and the 2 swapping mutants, GFP-tagged BCL2L1^{BCL2 BH3} or BCL2^{BCL2L1 BH3}. At 24 h post-transfection, the fixed cells were immunostained with an anti-MYC antibody (blue). Representative confocal microscopy images are shown. Scale bar: 10 μm. (E) Experiments were performed as described in (D), and the percentage of MYC-tagged FUNDC1-positive cells with LC3 puncta was quantified. Data represent mean ± s.e.m of 3 independent experiments (n > 100 cells per condition in each experiment). (F) HeLa cells were cotransfected with a MYC-tagged FUNDC1, a GFP vector or the 2 swapping mutants, GFP-tagged BCL2L1^{BCL2 BH3} or BCL2^{BCL2L1 BH3}. At 24 h post-transfection, cell lysates were prepared and the TOMM20, TIMM23, LC3, MYC, GFP, and ACTB protein levels were detected by western blot analysis. Grayscale values of the TOMM20, TIMM23, and LC3-II bands measured with ImageJ are shown under the corresponding bands to indicate the band intensities.