Fig 2: GAPDH enables CYP3A4 and 2D6 maturation to function. HEK293T cells that had been given scrambled or GAPDH-targeted siRNA were transfected to express FLAG and MYC-tagged CYP3A4 or 2D6 either alone or along with siRNA-resistant constructs of HA-tagged WT GAPDH or H53A GAPDH. A and B, quantification of the relative total GAPDH expression levels in the cells. C and D, CYP activities of the cell supernatants. Band intensities are from three independent experiments and the activity values are from two independent experiments of three replicates each. Significance designation ∗ p < 0.05, ns = not significant, one-way ANOVA.

Fig 3: Impact of PGRMC2 knockdown on cell heme allocation to CYP3A4 and 2D6. HEK293T cells were transfected with PGRMC2-directed siRNA for 24 h or given media alone and then were transfected to express FLAG and MYC-tagged CYP3A4 or 2D6 along with concurrent provision of 14C-δ-ALA + Fe-citrate, followed by harvest after an additional 8 h of culture. A and B, Representative Western blots indicating the relative expression levels of PGRMC2, CYP’s, and β-actin, and quantitative plots comparing the level of PGRMC2 expression under the various conditions. C, 14C counts in FLAG Ab pulldowns from supernatants of cells cultured under the indicated conditions. Band intensity and 14C-heme count values are from three independent experiments. Significance designation ∗ p < 0.05, ns = not significant, one-way ANOVA.

Fig 4: Effect of Hsp90 inhibitors on the functional maturation and heme content of CYP3A4 and 2D6. Heme-depleted HEK293T or GlyA-CHO cells were transfected to express FLAG- and MYC-tagged CYP3A4 or 2D6 and then given heme precursors without or with the Hsp90 inhibitors radicicol, ganetespib, or AUY922, and then cells were harvested after a further 6 to 8 h of culture. A and B, CYP activities measured in HEK293T cell supernatants for each condition. C and D, 14C counts in CYP antibody pulldowns from GlyA-CHO cell supernatants for each condition. Activity values are from two independent experiments of three replicates each. 14C heme count values are from three independent experiments. Significance designation ∗ p < 0.05, ns = not significant, one-way ANOVA.

Fig 5: Intracellular localization of CYP3A4 and 2D6 after transient expression in HEK293T cells. FLAG and MYC-tagged CYP3A4 or 2D6 were transiently expressed in HEK293T cells. A, cell supernatant, microsomal fraction, and supernatant minus microsome samples (50 μg protein each) were run on SDS-PAGE followed by Western blot analysis for the presence of the ER protein Calnexin and for FLAG-tagged CYP in each sample. B, cells expressing either CYP3A4 or 2D6 protein were processed for confocal microsocopy as described in Experimental Procedures. The extent of Calnexin and CYP (FLAG) colocalization is indicated by the orange color in the merged panels.