Journal: Cell Death & Disease

Article Title: BAG3 promotes autophagy and glutaminolysis via stabilizing glutaminase

doi: 10.1038/s41419-019-1504-6

Figure Lengend Snippet: a HepG2 or MCF7 cells were infected with lentivirus containing empty or BAG3 construct. Total protein was extracted and protein expression of indicated autophagy-related genes were investigated by immunoblotting. b HepG2 or MCF7 cells infected with lentivirus containing empty or BAG3 construct were treated with vehicle, CQ and E64D plus pepstatin A respectively, protein expression levels of LC3, p62 and BAG3 were analyzed using Western blot analysis. c HepG2 or MCF7 cells stably overexpressing EGFP-LC3B were infected with lentivirus containing empty or BAG3 construct. Cells were treated with vehicle, CQ or E64D plus pepstatin A, and the punctate distribution of EGFP-LC3B was visualized under the florescence microscopy. d Quantitative analysis of ( c ). Results shown represent mean ± SD from five representative microscopic fields and three independent experiments were performed. e HepG2 or MCF7 cells transduced with lentivirus containing empty or BAG3 construct, and ultrastructure was analyzed using transmission electron microscopy. * P < 0.05. Error bars indicate means ± SD

Article Snippet: Antibodies were used against the following: GLS (Sigma-Aldrich, WH0002744M1), GLS (Abcam, ab156876), GAPDH (Merck-Millipore, AB2302), LC3B (Origene, am20213pu), P62 (BD Biosciences, 610833), Beclin1 (Cell Signaling Technology, 3495), ATG3 (Cell Signaling Technology, 3415), ATG5 (Cell Signaling Technology, 12994), ATG7 (Cell Signaling Technology, 8558), ATG12 (Cell Signaling Technology, 4180), SIRT5 (Cell Signaling Technology, 8779), c-Myc (Invitrogen, R950-25), DYKDDDDK Tag (Cell Signaling Technology, 14793), HA-Tag (Cell Signaling Technology, 3724), pan-succinylation (PTM Biolabs, Hangzhou).

Techniques: Infection, Construct, Expressing, Western Blot, Stable Transfection, Microscopy, Transduction, Transmission Assay, Electron Microscopy

Journal: Cell Death & Disease

Article Title: BAG3 promotes autophagy and glutaminolysis via stabilizing glutaminase

doi: 10.1038/s41419-019-1504-6

Figure Lengend Snippet: a Control or BAG3-overexpressing HepG2 and MCF7 cells were transfected with scramble shRNA or shRNA specific against Beclin1 (shBeclin1), and Western blot analysis was performed using the indicated antibodies. b HepG2 or MCF7 cells transduced with lentivirus containing empty or BAG3 construct were treated with vehicle, 3-MA or WM respectively, protein expression levels of LC3, p62 and BAG3 were analyzed using Western blot analysis. c HepG2 or MCF7 cells stably overexpressing EGFP-LC3B were infected with lentivirus containing empty or BAG3 construct. Then cells were treated with vehicle, 3-MA or WM, and the punctate distribution of EGFP-LC3B was visualized under the florescence microscopy. d Quantitative analysis of ( c ). Results shown represent mean ± SD from five representative microscopic fields and three independent experiments were performed. * P < 0.05. N.S. not significant. Error bars indicate means ± SD

Article Snippet: Antibodies were used against the following: GLS (Sigma-Aldrich, WH0002744M1), GLS (Abcam, ab156876), GAPDH (Merck-Millipore, AB2302), LC3B (Origene, am20213pu), P62 (BD Biosciences, 610833), Beclin1 (Cell Signaling Technology, 3495), ATG3 (Cell Signaling Technology, 3415), ATG5 (Cell Signaling Technology, 12994), ATG7 (Cell Signaling Technology, 8558), ATG12 (Cell Signaling Technology, 4180), SIRT5 (Cell Signaling Technology, 8779), c-Myc (Invitrogen, R950-25), DYKDDDDK Tag (Cell Signaling Technology, 14793), HA-Tag (Cell Signaling Technology, 3724), pan-succinylation (PTM Biolabs, Hangzhou).

Techniques: Control, Transfection, shRNA, Western Blot, Transduction, Construct, Expressing, Stable Transfection, Infection, Microscopy