Fig. 4.

Cholesterol-Resorufin probe detects CYP11A1 activity. A, The enzymatic activity of CYP11A1 on the cholesterol-resorufin probe results in the production of fluorescent resorufin and pregnenolone. B, MA-10 cells incubated with 5 μm of the cholesterol resorufin probe for 24 h, after which the addition of cAMP results in a time-dependent increase in fluorescence; measurement was at 530ex/595em at the stated time points. C, Isolated mitochondria were incubated with 5 μm cholesterol-resorufin probe or cholesterol-resorufin probe and CYP11A1 inhibitor aminoglutethimide (AG). Measurement at 530ex/595em at stated time points demonstrate an increase in fluorescence in mitochondria not incubated with AG. D, Cholesterol-resorufin probe (50 μm) was incubated in control and hCG-stimulated gel slices. After addition of isocitrate to stimulate steroid production, CYP11A1 activity was measured at 530ex/595em, demonstrating increased activity at the 800-kDa complexes upon hCG treatment. E, Transiently transfected MA-10 cells were imaged 48 h after transfection of DsRed-ANT, CFP-CYP11A1 pair; DsRed-ANT, CFP-VDAC pair; or DsRed-TSPO, CFP-VDAC pair. Cells were treated for 2 h with dbcAMP and imaged. Mann-Whitney U test was performed to determine statistical differences of the E% between samples obtained before and after cAMP treatment. Results shown are means ± sem (n = 3); *, P < 0.05 **, P < 0.01.