Heme Regulation of Cellular Metabolism

Hemin, protoporphyrin IX, oligomycin A, carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), rotenone, antimycin A, 3-isobutyl-1-methylxanthine (IBMX), BSA, mannitol, norepinephrine, isoproterenol, 8-Br-cAMP, and succinylacetone were purchased from Sigma-Aldrich. CL 316,243 was obtained from Cayman Chemical. Forskolin was obtained from Chem Impex International. Insulin (Novolin) was purchased from Novo-Nordisk. Complete EDTA-free protease inhibitor cocktail was obtained from Roche. DMEM and other Gibco-branded cell culture products were purchased from Thermo Fisher. Heme-depleted FBS was prepared by treating FBS with 20 mM ascorbic acid for 16 hr, followed by 24 hr dialysis against PBS. Heme depletion was verified by measuring optical absorbance at 405 nm. CPAG-1 was synthesized as previously reported^{5 (link)}. ON-TARGET siRNA SMARTpools against human PGRMC2 (L-010639-00-0005), PGRMC1 (L-010642-00-0005), and mouse Nr1d1 (L-051721-00-0005), and BACH1 (L-042956-01-0005), as well as a Non-targeting Pool (D-001810-10-05) were purchased from Dharmacon. HEK293T cells were obtained from ATCC (CRL-3216) having undergone short-tandem repeat verification. Cells were routinely tested for mycoplasma and were never positive.