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. 2019 Aug 2;116(34):16997–17006. doi: 10.1073/pnas.1900748116

Fig. 2.

Fig. 2.

BIM-EL protein regulation by EglN3 is dependent on pVHL. (A) Anti–BIM-EL immunoblot of VHL+/+ and VHLfl/fl MEFs (immortalized by K1 large T antigen) that were transiently transduced with adenovirus encoding Cre-recombinase (Ad-Cre) or GFP control. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Anti–BIM-EL immunoblot analysis of 786-O VHL-null cells (−/−) stably transfected to generate WT VHL. (C) Immunoblot of 786-O cells with the indicated VHL status transduced with lentiviral pLKO shRNA targeting EglN3 or nontargeting control (shSCR). (D) Immunoblot of 786-O WT-VHL and VHL-null cells (−/−) under normoxic or anoxic conditions for 16 h. (E) Immunoblot of 786-O cells stably transfected to generate the indicated VHL species. (F, Left) Crystal violet staining of 786-O cells with the indicated VHL status. Cells were transiently transduced for 48 h with adenovirus (Adeno) encoding either WT EglN3 or GFP control. Cells were maintained in culture for an additional week before being subjected to crystal violet staining. (F, Right) Corresponding immunoblot is shown 48 h after infection. cl, cleaved. (G) Crystal violet staining and immunoblot of 786-O cells expressing HA-VHL that were stably transduced with lentiviral-encoded shRNA targeting BIM (shBIM54) or nontargeting control (shSCR). Cells were transiently transduced with adenovirus encoding EglN3 (Ad-E3) or GFP control. (H) Immunoblot analysis of stable polyclonal PC12 cells expressing the indicated human VHL (huVHL) species. PC12 clones were transiently transduced for 72 h with lentivirus encoding shRNA targeting endogenous rat VHL (endg. sh-ratVHL) or scramble control (shSCR). (Note: VHL long-term knockdown was not tolerated in PC12 cells.) (I) Percentage of fragmented nuclei of stable PC12 clones 3 d after cotransfection to generate GFP-histone and Flag-EglN3 along with pLKO plasmid targeting rat VHL (sh-rVHL) or scramble control (sh-SCR). One-way ANOVA was used to determine the statistical significance using GraphPad Prism software (****P < 0.0001). (J) Anti–BIM-EL immunoblot analysis of human normal adrenal glands and human primary PCC and PGL tumors. VHL mutation and 1p36 status have been characterized as indicated.