Cortisol Secretion Pathway Figure 2. Effects of ACTH and 8CPT-29-OMe-cAMP on Expression of Genes Coding for Steroidogenic Proteins. AZF cells were incubated either without (control), or with 8CPT-29-OMe-cAMP, 8CPT-29-OMe-cAMP plus H-89, or ACTH as indicated. Total RNA was isolated at indicated times. Each lane contained 10 mg of total RNA. Membranes were hybridized with specific probe for bovine CYP11a1, then stripped and probed for CYP17, stripped and reprobed for CYP21, as indicated. 18S rRNA bands from representative gels are shown as evidence of even loading. A) Concentration-dependent effect of 8CPT-29-OMe-cAMP on CYP11a1, CYP17, and CYP21 mRNA. AZF cells were either untreated (control, white bar) or treated with 10–50 mM 8CPT- 29-OMe-cAMP (grey bars) for 48 hr before isolating total RNA. B) Time-dependent effect of 8CPT-29-OMe-cAMP on CYP11a1, CYP17, and CYP21. AZF cells were incubated without (control, white bar), or with 8CPT-29-OMe-cAMP (30 mM) (grey bars), or pre-incubated with H-89 (10 mM) for 30 min before adding 8CPT-29-OMe-cAMP (30 mM) (striped/grey bar) for times indicated, after which total RNA was isolated. C) Comparison of time-dependent effects of 8CPT- 29-OMe-cAMP (30 mM) and ACTH (2 nM) on CYP17 mRNA. AZF cells were incubated either without (control, white bar) or with 8CPT-29-OMe-cAMP (30 mM, grey bar) or ACTH (2 nM, black bar) for 0.5 to 4 h (left panel) or 5 to 30 h (right panel), after which total RNA was isolated. doi:10.1371/journal.pone.0006088.g002 PLoS ONE | www.plosone.org 5 June 2009 | Volume 4 | Issue 6 | e6088