Fig. 3

CYP19A1 regulates mitochondrial function depending on its enzymatic activity and estrogen biosynthesis. (A) The in vitro enzymatic activity of wild-type (WT) CYP19A1 and its mutants, D309N and Y361F, was determined using an activity assay. (B-F) CYP19A1 knockout SW480 and HT29 cells were transduced with lentiviral vectors to express exogenous WT, D309N, or Y361F CYP19A1, generating four cell lines: KO, KO + WT, KO + D309N, and KO + Y361F. (B) Relative estrogen levels were measured in WT, KO, KO + WT, KO + D309N, and KO + Y361F cells using a commercially available assay kit. (C-F) The OCR of KO, KO + WT, KO + D309N, and KO + Y361F cells was measured using the Seahorse Mito Stress Test. Basal, ATP-linked, and maximal respiration were assessed by sequential treatment with oligomycin (O), FCCP (F), and rotenone/antimycin A (R/A). (G-J) The OCR of wild-type and CYP19A1 knockout SW480 and HT29 cells with or without estradiol (E2) treatment was measured using the Seahorse Mito Stress Test. Statistical significance was determined using Student’s t-test according to the indicated comparisons in each panel (* p < 0.05, ** p < 0.01, *** p < 0.001, ns: not significant). Data are presented as mean ± SD. (A, B) n = 3; (C-J) n = 6