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. 2023 Jan 17;133(2):e163498. doi: 10.1172/JCI163498

Figure 2. BMX is required for DHEA metabolism by 3βHSD1.

Figure 2

(A) LNCaP cells were treated with ibrutinib or zanubrutinib for 1 hour and subsequently treated with [3H]-DHEA for 5 hours, followed by steroid extraction from media and steroid separation and quantitation with HPLC. The experiment was done in triplicate and repeated in independent experiments. Mean ± SEM represents 3 replicates in 1 experiment. 3 independent experiments were performed. **P < 0.01, ***P < 0.001, ****P < 0.0001 (1-way ANOVA test with Dunnett’s multiple comparisons test). (B) Cells stably expressing shNT or 2 shRNA sequences against BMX were treated with [3H]-DHEA for 6 hours and analyzed as in (A). Mean ± SEM represents combined data from 3 biological independent replicates performed in technical triplicate. ***P < 0.001, ****P < 0.0001 (1-way ANOVA test with Dunnett’s multiple comparisons test). (C) 293T cells were transiently cotransfected with HA-BMX, EGFR, SRC, or YES and GST-3βHSD1, followed by GST pull-down and Western blot (left). 293T cells were transiently cotransfected with HA-BMX and GST-3βHSD1, followed by HA immunoprecipitation and Western blot (right). (D) LNCaP and C4-2 cells were cultured, protein collected, and immunoprecipitation and Western blot performed for endogenously expressed proteins. WCL blots run in parallel, contemporaneously, using identical samples are shown. (E) LNCaP cells were transiently cotransfected with HA-BMX, and GST-3βHSD1cells were starved with medium containing 10% charcoal-stripped FBS for 24 hours, then treated with steroids for 2 hours, followed by GST-pull down and Western blot to detect interaction of HA-BMX and GST-3βHSD1. (F) LNCaP cells were starved as in (E), then transfected with HA-BMX and treated with steroids for 2 hours; p-BMX was detected by Western blot. Blots run in parallel, contemporaneously, using identical samples are shown. (G) Stable C4-2 cell lines with HSD3B1 gRNA or control gRNA were transfected with HA-BMX, starved as in (E) and treated with DHEA for 2 hours; p-BMX was detected by Western blot.