Fig 3. Effect of MAMLD1 on CYP17A1 promoter and enzyme activities.

HEK293 cells or NCI-H295R cells were transiently transfected with MAMLD1 WT and mutant expression vectors. For promoter activation studies, the (-3.7kb) CYP17A1 promoter luciferase reporter construct was co-transfected. A. CYP17A1 promoter activation by MAMLD1 was assessed by the Promega Dual luciferase assay in HEK293 cells. Only for mutant MAMLD1 L210X and L724V an impaired CYP17A1 activation was found. Results are expressed in RLU and represent the mean and SEM of 3 independent experiments performed in duplicate. B. The effect of WT and mutant MAMLD1 on CYP17A1 enzyme activity was assessed in transfected NCI-H295R, MA-10 and HEK293 cells by measuring the conversion of progesterone to 17-hydroxyprogesterone. Steroid production was labeled with [¹⁴C]progesterone for 60 min. Steroids were extracted and resolved by thin-layer chromatography, then quantified as % conversion. A representative steroid profile obtained from NCI-H295R cells is shown (n = 2). No effect of MAMLD1 on CYP17A1-hydroxylase activity was detected. P: progesterone; 17OHP: 17-hydroxyprogesterone; RLU: relative light units; Ve: empty vector; WT: wild type; NT: non-transfected; * p≤0.05.