Figure 4. ARL6IP1 regulates mitophagy... | Rockefeller University Press

Figure 4.

$ARL6IP1 regulates mitophagy through interaction with LC3B and BCL2L13 in autophagosome formation. (A) Immunofluorescence detection of LC3B-II puncta in HeLa cells transfected with mCherry-empty vectors or mCherry-ARL6IP1 after 20 μM CCCP and/or 10 μM wortmannin treatment for 6 h (left, 600×; scale bar: 20 μm). Quantification of LC3B-II puncta per cell in HeLa cells stably expressing GFP-LC3B (right). Data presented as mean values of triplicates ± SD (≥10 images assessed per group). **P < 0.01. (B) Protein levels of autophagosome-related genes were analyzed using western blotting in ARL6IP1 WT and KO MEFs at three passages after 10 µM CCCP treatment for 6 h. (C) Live-cell fluorescence image of HeLa cells cotransfected with indicated vectors (400×; scale bar: 50 μm) observed under a fluorescence microscope and magnified in a bright field (600×; scale bar: 20 μm). (D) Interaction of endogenous ARL6IP1, LC3B, and p62 by IP-western blotting in ARL6IP1 KO HEK293T cells via CRISPR/Cas9-mediated gene editing. (E) Interaction of ARL6IP1 and BCL2L13 by IP-western blotting in HEK293T cells. (F)ARL6IP1 KO and WT MEFs were cultured in starvation medium (HBSS) for 1 h. After flotation assay in iodixanol gradients, fractions were detected by western blotting. Asterisks indicate the floatation of omegasome-related proteins from ARL6IP1 KO and WT MEFs. The arrow indicates the position of the ATG9A protein. Data represent averages of three independent biological replicates. Source data are available for this figure: SourceData F4.$

ARL6IP1 regulates mitophagy through interaction with LC3B and BCL2L13 in autophagosome formation. (A) Immunofluorescence detection of LC3B-II puncta in HeLa cells transfected with mCherry-empty vectors or mCherry-ARL6IP1 after 20 μM CCCP and/or 10 μM wortmannin treatment for 6 h (left, 600×; scale bar: 20 μm). Quantification of LC3B-II puncta per cell in HeLa cells stably expressing GFP-LC3B (right). Data presented as mean values of triplicates ± SD (≥10 images assessed per group). **P < 0.01. (B) Protein levels of autophagosome-related genes were analyzed using western blotting in ARL6IP1 WT and KO MEFs at three passages after 10 µM CCCP treatment for 6 h. (C) Live-cell fluorescence image of HeLa cells cotransfected with indicated vectors (400×; scale bar: 50 μm) observed under a fluorescence microscope and magnified in a bright field (600×; scale bar: 20 μm). (D) Interaction of endogenous ARL6IP1, LC3B, and p62 by IP-western blotting in ARL6IP1 KO HEK293T cells via CRISPR/Cas9-mediated gene editing. (E) Interaction of ARL6IP1 and BCL2L13 by IP-western blotting in HEK293T cells. (F)ARL6IP1 KO and WT MEFs were cultured in starvation medium (HBSS) for 1 h. After flotation assay in iodixanol gradients, fractions were detected by western blotting. Asterisks indicate the floatation of omegasome-related proteins from ARL6IP1 KO and WT MEFs. The arrow indicates the position of the ATG9A protein. Data represent averages of three independent biological replicates. Source data are available for this figure: SourceData F4.