Journal: Science Advances

Article Title: BAP31 regulates mitochondrial function via interaction with Tom40 within ER-mitochondria contact sites

doi: 10.1126/sciadv.aaw1386

Figure Lengend Snippet: ( A ) U2OS cells subjected to BAP31 knockout via CRISPR-Cas9 system (sgControl and sgBAP31-2) were subjected to TEM. Higher magnification images are shown at the right. Scale bars, 10 and 1 μm (left and right, respectively). White arrows indicate autolysosomes. ( B ) Total number of autolysosomes in each cell was determined. Data are presented as means ± SD ( n = 6). ( C ) Loss of BAP31 increases LC3-II expression. U2OS cells were transfected with the indicated concentrations of siBAP31 and 150 pmol of siControl for 24 hours. Cells were subjected to immunoblotting using anti-BAP31, anti-LC3, and anti–β-actin antibodies. ( D ) U2OS cells stably expressing GFP-LC3 were transfected with 100 pmol of siBAP31 or siControl for 24 hours. Cells were fixed with 4% paraformaldehyde, and GFP-LC3 (green) fluorescence was determined. Blue represents nuclear 4′,6-diamidino-2-phenylindole (DAPI) staining. Scale bar, 10 μm. The number of LC3-GFP puncta in the cells (green dots) was determined, and data are presented as means ± SD ( n = 6). ( E ) Loss of BAP31 stimulates autophagosome synthesis. U2OS cells were transfected with 100 pmol of siControl or siBAP31 for 24 hours, followed by treatment with or without bafilomycin A1 (1 μg/ml) for 1 hour. Cells were subjected to immunoblotting using the indicated antibodies. ( F ) BAP31 does not affect the ER stress response. U2OS cells were transfected with siBAP31 and siControl for 18 hours and then treated with or without BFA (1 μg/ml) for 8 hours. Cells were subjected to immunoblotting using the indicated antibodies and Phos-tag SDS–polyacrylamide gel electrophoresis (PAGE) or normal SDS-PAGE. ( G ) BAP31 knockout or knockdown activates the AMPK-ULK-LC3 signaling pathway. U2OS and HeLa cells subjected to BAP31 knockout via the CRISPR-Cas9 system (sgControl, sgBAP31-2, and sgBAP31-3) and MEF cells transfected with siControl (200 pmol) and siBAP31 at the indicated concentrations for 24 hours were subjected to immunoblotting using the indicated antibodies. P value was calculated using two-way analysis of variance (ANOVA). ** P < 0.01 (B and D).

Article Snippet: Antibodies used for immunoblotting were specific for the following proteins: LC3b, PERK, IRE1α, P-AMPK, AMPK (total), P-ULK (Ser 757 ), P-ULK (Ser 317 ), ULK (total), COXIV, cytochrome c, calnexin, HSP60, mitofusin-1, and Bcl-2 (Cell Signaling Technology); BAP31, ATF6, Tom40, Tom22, prohibitin, and VDAC1 (Santa Cruz Biotechnology); Parkin and FACL-4 (Abcam); and NDUFS4, NDUFB11, α-tubulin, β-actin, and flag (Sigma-Aldrich).

Techniques: Knock-Out, CRISPR, Expressing, Transfection, Western Blot, Stable Transfection, Fluorescence, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Knockdown