Journal: Molecular and Cellular Biology

Article Title: Increased O-GlcNAcylation of SNAP29 Drives Arsenic-Induced Autophagic Dysfunction

doi: 10.1128/MCB.00595-17

Figure Lengend Snippet: Arsenic inhibits autophagic flux by blocking autophagosome-lysosome fusion. (A) HeLa cells were left untreated or treated with 0.5, 1, or 2 μM sodium arsenite [As(III)] for 4 h. Immunoblot analysis of SQSTM1 and MAP1LC3A/B-I and MAP1LC3A/B-II protein levels was performed. (B) HeLa cells were left untreated or treated with 0.5, 1, or 2 μM As(III) or 100 nM bafilomycin A1 (BAF) for 4 h. mRNA levels were measured using quantitative real time-PCR. β-Actin was used as an internal control. (C) iBMKs stably expressing GFP-LC3 were either left untreated or treated with 1× Hanks' balanced salt solution (HBSS; Starve) or 2 μM As(III) for 4 h. Immunoblot analysis of free GFP levels was then performed. (D) NIH 3T3 cells were treated with 1 or 2 μM As(III), HBSS, 100 nM BAF, or As(III) plus BAF or HBSS plus BAF. Immunoblot analysis of MAP1LC3A/B-I and MAP1LC3A/B-II protein levels was performed. Actin and GAPDH were used as internal loading controls. Data are means ± SEMs (n = 3). *, P < 0.05 compared to the value for the control (Student's t test). (E) NIH 3T3 cells were treated with 2 μM As(III), 100 nM BAF, or 40 μM chloroquine (CQ) for 4 h. Following treatment, lysosomal pH was detected using LysoSensor Yellow/Blue DND-160. Blue to yellow/white indicates an increase in lysosomal pH. Scale bar = 10 μm. (F) NIH 3T3 cells were transfected with 1 μg of the mRFP-GFP-LC3 tandem fluorescent reporter and 1 μg of LAMP1-CFP for 24 h and left untreated or treated with 2 μM As(III), HBSS, or 100 nM BAF for 4 h. Cells were subjected to immunofluorescence analysis to detect autophagosomes (yellow), lysosomes (blue), and autolysosomes (pink). mRFP-GFP-positive puncta and percent autolysosomes were determined by quantifying puncta from five cells, and repeating three times. Data are means ± SEMs (n = 5). *, P < 0.05 compared to the value for the control (Student's t test). Scale bar = 10 μm.

Article Snippet: The primary antibody against green fluorescent protein (GFP; 42060) was purchased from GeneTex.

Techniques: Blocking Assay, Western Blot, Real-time Polymerase Chain Reaction, Control, Stable Transfection, Expressing, Transfection, Immunofluorescence