lysosomal inhibitor bafa1  (MedChemExpress)

Journal: The EMBO Journal

Article Title: Intrinsic lipid binding activity of ATG 16L1 supports efficient membrane anchoring and autophagy

doi: 10.15252/embj.2018100554

Figure Lengend Snippet: ATG16L1 −/− cells were reconstituted with ATG16L1 WT ‐ or ATG16L1 LD ‐expression constructs and autophagy assessed during treatment with various stimuli followed by immunoblotting using the indicated antibodies. Cells were amino acid starved (AA starve) to induce mTORC1‐dependent autophagy in the presence or absence of BafA1 for 2 h prior to lysis. Mitophagy was induced by CCCP (10 μM) treatment for 6 h prior to lysis. Glucose starvation (Glu starve) for 20 h was used to induce mTORC1‐independent autophagy. BafA1 was added to all conditions 2 h prior to lysis. LC3‐associated phagocytosis (LAP)‐like LC3 lipidation was induced by treating cells with CCCP (100 μM) for 2 h prior to lysis. Right panel shows quantification of LC3‐II/LC3‐I ratio. Means and error bars (SEM) are shown ( n = 4). ** P ≤ 0.01 (pairwise unpaired Student's t ‐test). LAP‐like LC3 lipidation induced by monensin or ammonium chloride (NH 4 Cl) for 2 h prior to lysis.

Article Snippet: Bafilomycin A1 (BafA1, inhibitor of lysosome acidification) was purchased from Sigma or Tocris and used at a final concentration of 20 nM.

Techniques: Expressing, Construct, Western Blot, Lysis

Journal: The EMBO Journal

Article Title: Intrinsic lipid binding activity of ATG 16L1 supports efficient membrane anchoring and autophagy

doi: 10.15252/embj.2018100554

Figure Lengend Snippet: Protein–protein interaction assay in 293T cells transiently transfected with the indicated Flag‐S‐tagged ATG16L1 constructs and GFP‐Rab33B. S tag pull‐down was performed, and protein complexes were analysed by immunoblotting using the indicated antibodies. Wild‐type MEFs or Rab33B −/− clones were amino acid starved (AA starve) to induce autophagy in the presence of BafA1 for 2 h prior to lysis. Cell lysates were analysed using the indicated antibodies. Cells as in (B) were amino acid starved for 2 h prior to immunofluorescence analyses using antibodies against endogenous ATG16L1 (red) and WIPI2 (green). Quantification of ATG16L1 positive puncta is shown on the right panel depicting means and error bars (SEM) from at least three independent experiments. ns P > 0.05 (pairwise unpaired Student's t ‐test). Scale bar: 9 μm.

Article Snippet: Bafilomycin A1 (BafA1, inhibitor of lysosome acidification) was purchased from Sigma or Tocris and used at a final concentration of 20 nM.

Techniques: Protein Protein Interaction Assay, Transfection, Construct, Western Blot, Clone Assay, Lysis, Immunofluorescence

Journal: The EMBO Journal

Article Title: Intrinsic lipid binding activity of ATG 16L1 supports efficient membrane anchoring and autophagy

doi: 10.15252/embj.2018100554

Figure Lengend Snippet: ATG16L1 −/− cells were reconstituted with ATG16L1 WT ‐ or ATG16L1 LE ‐expression constructs and autophagy assessed during amino acid starvation (AA starve) followed by immunoblotting or immunofluorescence analyses. Immunofluorescence analyses of amino acid starved cells (2 h) using antibodies against WIPI2 (red) or ATG16L1 (green). Scale bar: 9 μm. Cells as in (A) with the addition of 3′MA (and pretreatment with the drug for 30 min). Scale bar: 9 μm. Quantification of percentage of cells positive for ATG16L1 puncta in (A) and (B). Cells were left untreated, amino acid starved for 2 h or amino acid starved for 2 h followed by refeeding with full growth media for 1 h. Puncta formation was assessed by immunofluorescence staining using antibodies against S tag to detect ATG16L1. Scale bar: 10 μm. Immunoblot analyses of amino acid starved cells in the presence or absence of BafA1 for 2 h. Cell lysates were analysed using the indicated antibodies. WIPI2 −/− cells reconstituted with the indicated ATG16L1 constructs were amino acid starved for 2 h. BafA1 treatment was included in all conditions for 2 h prior to lysis. Cell lysates were analysed by immunoblotting using the indicated antibodies. Quantification of LC3‐II/LC3‐I ratio of three independent experiments is shown on the right panel. Ferroptosis assay in ATG16L1 −/− cells stably expressing ATG16L1 WT , ATG16L1 LD or ATG16L1 LE . Cells were cultured in amino acid free media in the presence of 10% FBS or 10% dialysed FBS (diFBS) for 24 h. Quantification of percentage of PI‐positive cells from at least three independent experiments is shown. Data information: Quantifications depict means and error bars (SEM) from at least three independent experiments. ns P > 0.05, * P ≤ 0.05, ** P ≤ 0.01 (pairwise unpaired Student's t ‐test).

Article Snippet: Bafilomycin A1 (BafA1, inhibitor of lysosome acidification) was purchased from Sigma or Tocris and used at a final concentration of 20 nM.

Techniques: Expressing, Construct, Western Blot, Immunofluorescence, Staining, Lysis, Stable Transfection, Cell Culture