Fig. 3

BAG1S mediated cell survival dependent on interaction with HSP70 chaperone. a Schematic demonstrating BAG1S interaction with HSP70 chaperone (top) and a mutant BAG1ΔS that retains BAG1S structure but prevents HSP70 binding via a single amino acid change in the BAG domain (bottom). b U2OS MYC-ER cells transfected with vectors encoding BAG1S, BAG1ΔS or empty vector, followed by endogenous BAG1 depletion or shLUC control. IB demonstrating efficient knockdown of endogenous BAG1 and retained ectopic BAG1S and BAG1ΔS expression. c U2OS MYC-ER cells expressing ectopic vector, BAG1S, or BAG1ΔS depleted of endogenous BAG1 via lentiviral infection. Five days post-infection cells treated with ±100 nM 4-OHT for 48 h. Apoptosis measured by Annexin V-PE plus 7AAD DNA staining and quantified by FACS analysis. d Quantification of three experimental replicates representing average and standard deviation of cumulative early and late apoptosis based on population of Annexin V-PE positive cells. e PARP cleavage and BAG1 knockdown demonstrated by IB. F and C indicate full and cleaved species respectively. f U2OS MYC-ER cells cultured with ±100 nM 4-OHT and 10uM MKT-077 HSP70 inhibitor for 48 h. CAS-3 demonstrated by IB. F and C indicate full and cleaved species respectively. g Model detailing three experimental methods used to interrupt HSP70/BAG1S chaperone complex activity. Limiting HSP70/BAG1S function via silencing BAG1, inhibiting HSP70, and preventing BAG1S binding HSP70 all resulted in increased MYC-dependent death