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. 2024 Jul 24;25(9):3896–3924. doi: 10.1038/s44319-024-00206-6

Figure EV5. (belonging to Fig. 7): Inhibition of BOK-mediated apoptosis by BCL-2 depends on BOK-TMD.

Figure EV5

(A) Detection of various Bcl-2 family proteins in whole cell lysates of HEK293FT and DU145 cells using Western blot. β-actin was used as loading control. One independent experiment. (B) HEK293FT cells were transfected with siRNA for BCL-2 (siBCL-2) or control siRNA (siCTRL) for 24 h and were subsequently transfected with plasmids for the expression of EGFP-BOK or EGFP. After 18 h, cells were harvested and analyzed by Western blot. PARP, EGFP(-BOK) and BCL-2 expression were detected. β-actin was used as loading control. Representative blot from two independent experiments. (C) HEK293FT cells were co-transfected with plasmids for the expression of a CRISPR/Cas9 vector targeting BCL2 and EGFP or EGFP-BOK (empty = empty vector backbone control). After 18 h, cell death was assessed using Annexin-V-APC staining and flow cytometry. EGFP+/Annexin-V+ cells were detected by flow cytometry. Mean ± sd from three independent experiments. *p < 0.05, one-way ANOVA with Tukey’s multiple comparison test. (D) DU145 cells were transfected with plasmids for the expression of EGFP-BOK or EGFP in combination with mCherry-BCL-2 or an empty vector as a control. After 42 h, cells were stained with Annexin-V-APC and cell death (EGFP+/Annexin-V+ cells) was assessed using flow cytometry. Mean ± sd from three (EGFP) or six (EGFP-BOK) independent experiments. ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparison test. (E) DU145 cells were transfected with plasmids for the expression of EGFP, EGFP-BOKBAX-TMD, or EGFP-BOKBAX-TMD L70E in combination with mCherry-BCL-2 or an empty vector as a control. After 18 h, cells were stained with Annexin-V-APC and cell death (EGFP+/Annexin-V+ cells) was assessed using flow cytometry. Mean ± sd from three independent experiments. (F) DU145 cells were transfected with plasmids for the expression of EGFP, EGFP-BOK, EGFP-BAX and EGFP-BOKBAX-TMD and incubated with 10 µM pan-caspase inhibitor QVD-OPh (+QVD) or DMSO as control (-QVD). After 18 h, cells were stained with Annexin-V-APC and cell death (EGFP+/Annexin-V+ cells) was assessed using flow cytometry. Mean ± sd from three independent experiments. (G, H) MCF-7 cells were transfected with plasmids for the expression of either EGFP-BOKBAX-TMD (G) or EGFP-BOKBAX-TMD L70E (H). Cells were either stained with Mitotracker red after 24 h or co-transfected to express mCherry-BCL-2. Twenty-four hours post transfection cells were fixed and analyzed by cLSM. Images are maximum projection of z-stacks representative of two independent experiments. Scale bar = 10 µm.