Fig. 3: BAG5 enhances cell death and causes increased PARP and Caspase-3 cleavage following CCCP treatment.

a Dose–response curve for CCCP in the GFP and GFP-BAG5 stable cell lines using PrestoBlue viability assay. Experiment performed three times in triplicate. Data back normalized to 0 treatment condition, and statistical analysis was done using two-way ANOVA followed by Bonferroni post hoc testing (***p < 0.001). b Fold change in cell death elicited by 50 μm CCCP treatment (CCCP/DMSO) in the GFP and GFP-BAG5 SH-SY5Y stable cell lines as measured by the DRAQ7 cell death assay using flow cytometry. GFP and GFP-BAG5 expression induced for 24 h using 2 μg/mL doxycycline prior to treatment. Experiment performed two times in triplicate. Statistical significance determined by unpaired t test (****p < 0.0001). Columns represent mean ± SEM. c Same as b comparing the effect of BAG5 KD (siBAG5) to control (siNTC). d Same as b comparing the effect of Parkin KD (siParkin) to control (siNTC) in SH-SY5Y GFP-BAG5 cells. e Western blot demonstrating Parkin knockout (KO) in Parkin KO HEK293T cells compared with the wild-type (WT) HEK293T cells and relative levels of transfected GFP and GFP-BAG5. f Relative percentage of cell death elicited by 50 μm CCCP in Parkin KO HEK293T cells relative to WT HEK293T cells transfected with GFP-BAG5 or GFP alone as a control. Experiment performed two times in triplicate. Statistical analysis was done using one-way ANOVA with Tukey’s post hoc test (**p < 0.01, ***p < 0.001, ****p < 0.0001). Columns represent mean ± SEM. g Western blot illustrating the changes in endogenous PARP, cleaved PARP (cPARP), Caspase-3 (Casp3), and cleaved Caspase-3 (cCasp3) in the GFP and GFP-BAG5 SH-SY5Y stable cell lines following treatment with 50 μm CCCP. Representative blot from three independent experiments. h Quantification of the western blot presented in g. Data obtained from three independent experiments and statistical analysis done using one-way ANOVA followed by Bonferroni post hoc testing (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Columns represent mean ± SEM.