Journal: Cell Death and Differentiation

Article Title: AMBRA1 is able to induce mitophagy via LC3 binding, regardless of PARKIN and p62/SQSTM1

doi: 10.1038/cdd.2014.139

Figure Lengend Snippet: AMBRA1–ActA induces mitophagy in PARKIN-deficient cell lines. ( a ) HeLa and HEK293 cells were grown in normal media. After extraction of proteins, we performed a western blot analysis by using antibodies against PARKIN and against ACTIN (as a loading control). ( b ) Colocalization between AMBRA1–ActA, mito-RFP and LC3 protein. HeLa cells were co-transfected with vectors encoding AMBRA1–ActA and mito-RFP, and grown in normal media. Cells were then stained using antibodies anti-myc (AMBRA1, blue) and anti-LC3 (green). Nuclei were stained with 1 μ g/ μ l DAPI for 20 min. Merge of the different fluorescence signals is illustrated. Scale bar, 8 μ m. ( c ) Quantification in HeLa cells of AMBRA1–ActA/mito-RFP mito-colocalizations at 24, 48 and 72 h after transfection per cell (±S.D.). Each time point value is the mean±S.D. from three independent experiments. Statistical analysis was performed using Student's test (*** P <0.001; 24 h versus 72 h). ( d ) HeLa cells were transfected with a vector encoding myc-AMBRA1–ActA. At 24, 48 and 72 h after transfection, proteins extracts were analysed by western blot using the following antibodies: anti-MnSOD, anti-TOM20, anti-myc (to control AMBRA1–ActA expression) and anti-ACTIN (loading control). The graph represents the MnSOD/ACTIN ratio (±S.D.). Each point value represents the mean±S.D. from three independent experiments. Statistical analysis was performed using Student's test (** P <0.01; 24 h versus 72 h). ( e ) HEK293 cells were co-transfected with vectors encoding ShPARKIN or control ShRNA and myc-AMBRA1-ActA. At 24 h after transfection, expression of PARKIN was controlled by western blot analysis by means of anti-PARKIN and anti-ACTIN (loading control) antibodies. Cells were fixed and stained with an anti-myc antibody (green). The merge of the fluorescence signals is shown in the right panels. Scale bar, 4 μ m. The graph illustrates the quantification of AMBRA1–ActA/mito-RFP colocalizations on mito-aggresomes per cell (±S.D.). Each point value represents the mean±S.D. from two independent experiments. Statistical analysis was performed using Student's test (* P <0.01; 24 h versus 72 h). ( f ) HEK293 cells were co-transfected with vectors encoding ShPARKIN or control ShRNA, and myc-AMBRA1–ActA. At 24 h after transfection, expression of PARKIN was checked by western blot analysis by means of anti-PARKIN and anti-ACTIN (loading control) antibodies. At 24, 48 and 72 h after transfection, protein extracts were analysed by western blot using the following antibodies: anti-MnSOD, anti-TOM20, anti-myc (to control AMBRA1–ActA expression) and anti-ACTIN (loading control). The graph represents the MnSOD/ACTIN ratio (±S.D.). Each point value represents the mean±S.D. from three independent experiments. Statistical analysis was performed using Student's test (* P <0.05; 24 h versus 72 h). ( g ) HeLa cells were co-transfected with vectors encoding myc-AMBRA1–ActA and mito-RFP (red). At 24 h after transfection, cells were fixed and stained with anti-myc (AMBRA1, blue) and anti-p62 antibodies (green). Merge of the fluorescence signals are shown. N, nucleus. Scale bar, 4 μ m

Article Snippet: ShRNA PARKIN (PGIP2 449497) or shRNA Control (PGIP2 ) were transfected in HEK293 cells using Turbofect Transfection reagent (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA, R0531), according to the manufacturer's instructions.

Techniques: Extraction, Western Blot, Control, Transfection, Staining, Fluorescence, Plasmid Preparation, Expressing, shRNA