Fig 2: BAG3 decreases p53 activity under glucose insufficiency. (A-C), immunoblot analysis of p53 expression in HCT116 (A), MCF7 and Bel-7402 (B), and MEF (C) cells. D-E, Luciferase assays for p53 responsive construct in HCT116 (D) or MEF (E) cells. (F-G), Analysis of novel protein synthesis by HPG incorporation in HCT116 (F) or MEF (G) cells. H, immunoblot analysis of BAG3 expression in HCT116 cells. I, crystal violet assay for HCT116 cells

Fig 3: BAG3 suppresses cell viability under metabolic stress via direct interaction with p53. A, DuoLink PLA analysis of endogenous expression BAG3 and p53 interaction in HCT116, MCF7 and Bel-7402 cells. B, immunoblot analysis from immunoprecipitated Flag-tagged full-length (FL) or mutant BAG3 in HEK293 cells. C, immunoblot analysis from immunoprecipitated Myc-tagged FL or mutant p53 in HEK293 cells. D, immunoblot analysis of p53 in HCT116 cells transfected with FL or mutant BAG3. E, crystal violet assay for HCT116 cells transfected with FL or mutant BAG3

Fig 4: BAG3 promotes calpain-dependent p53 degradation under metabolic stress. A, real-time RT-PCR analysis of p53 mRNA in HCT116, MCF7 and Bel-7402 cells. B, immunoblot analysis of p53 in HCT116 and MEF cells cultured with 1 mmol/L glucose for the indicated time. C immunoblot analysis of p53 in the presence of protein degradation inhibitors (MG132, E64D and pepstatin A) or protein synthesis inhibitor cycloheximide (CHX) in the indicated cells. (D-F), HCT116 (D), MEF (E), MCF7 and Bel-7402 (F) cells were treated with 25 and 1 mmol/L glucose, immunoblot analyses were performed using the indicated antibodies. G, immunoblot analysis of p53 in HCT116 cells cultured under 1mM glucose in the presence of calpain inhibitor EDTA and Z-LLY-FMK

Fig 5: BAG3 reduction is a protective response of cells to glucose insufficiency. A, immunoblot analysis of ectopic BAG3 in HCT116 cells infected with lentivirus containing empty or BAG3 construct. B, viable cell count of HCT116 cells cultured under 1mM glucose. C, apoptotic cell analysis of HCT116 cells. D, immunoblot analyses of p53 and p21 protein in HCT116 cells. E, cell cycle analysis of HCT116 cells. F, immunoblot analysis of ectopic BAG3 in MCF7 and Bel-7402 cells. (G-H), viable cell count of MCF7 (G) and Bel-7402 (H) cells cultured under 1mM glucose. (I-J), cell cycle analysis of MCF7 (I) and Bel-7402 (J) cells. K, immunoblot analysis of BAG3 expression in control or BAG3 knock in (KI) MEF cells. L, crystal violet assay for MEF cells. M, cell cycle analysis of MEF cells