Fig 1.
Effects of 25 μM mitotane and 20 μM metyrapone on cortisol levels.
Four independent experiments were used to determine each data point. Metyrapone or mitotane treatment vs. untreated cells, P <0.05. Combined treatment with mitotane and metyrapone vs. untreated cells. P = 0.005.
Fig 2.
Effects of mitotane and 20 μM metyrapone on H295R cell viability.
Three technical replicate wells for each experiment (n = 3) were used to determine each data point. P = ns comparing mitotane treatment vs. combined treatment with mitotane and metyrapone.
Fig 3.
Mitotane metabolites in ACC cells: Uptake, bioavailability and metabolization.
Intracellular and extracellular concentration of mitotane and its metabolites in H295R, measured by means of HPLC–UV after metyrapone treatment. Six independent experiments were used to determinate each data point.
Fig 4.
CYP11B1 expression after treatment with mitotane and metyrapone, or both.
Data obtained from two independent experiments with two replicates for each experiment, and expressed as fold changes compared to β-actin expression (2-ΔΔCt). A > 2 fold increase was considered significant.
Fig 5.
Efficiency of siRna transfection and silencing.
(A) H295R cells transfected with a fluorescent siRna indicator. (B) CYP11B1 gene expression in CYP11B1 siRna cells and non-targeting siRna cells. Data obtained from two independent experiments with two replicates for each experiment. (C) Cortisol levels in CYP11B1 siRna cells and non-targeting siRna control cells. Four independent experiments were used to determine each data point.
Fig 6.
Mitotane cytotoxicity and metabolism after CYP11B1 modulation in H295R cell line.
(A) Mitotane cytotoxicity was not influenced by CYP11B1 interference (p = ns, comparing IC50 doses of CYP11B1 siRna cells and non-targeting siRna control cells). Three replicate wells for each experiment (n = 2) were used to determine each data point. (B) CYP11B1 modulation did not influence uptake of mitotane and its metabolization (p = ns, comparing drug levels of CYP11B1 siRna cells and non-targeting siRna control cells). Four independent experiments were used to determinate each data point, measured in HPLC-UV.