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. 2018 Nov 21;115(50):12781–12786. doi: 10.1073/pnas.1803826115

Fig. 4.

Fig. 4.

SdY prevents the Foxl2/Nr5a1 positive regulation of the cyp19a1a promoter. Medaka cyp19a1a promoter activity (pGL3-cyp19a1a promoter coupled to a firefly luciferase) was measured using a luciferase reporter assay after HEK 293T cell cotransfection with either medaka nr5a1 and/or foxl2 expression plasmids and variable concentrations of the rainbow trout sdY expression plasmid. All results were calculated as the mean ± SEM of three biological replicates in two independent experiments. (A) Foxl2 and Nr5a1 act in synergy to induce cyp19a1a expression. Medaka cyp19a1a luciferase assay with variable concentrations of foxl2 (50–400 ng) and nr5a1 (50–200 ng) or 100 ng of nr5a1 with variable concentrations of foxl2 (50–400 ng). Statistical significances of luciferase activity within treatments were tested using an unpaired two-tailed Student’s t test. Effects of foxl2 alone and nr5a1 alone compared with their synergistic effect (shown by asterisks on the dotted lines joining the different groups) were tested by a one-way ANOVA with a post hoc Dunnett test. (B) SdY prevents Foxl2/Nr5a1 positive regulation of the cyp19a1a promoter. Medaka cyp19a1a luciferase assay with variable concentrations of foxl2 (50–400 ng) combined with a fixed concentration of nr5a1 (100 ng) and a fixed concentration of foxl2 (200 ng) and nr5a1 (100 ng) combined with variable concentrations of sdY (25–300 ng). Empty plasmid control (pGL3) and Foxl2 alone (200 ng) are depicted by a + sign. Statistical significances of luciferase activity were tested using a Mann–Whitney U test. (C) SdY does not repress cyp19a1a promoter expression induced by medaka Foxl2 alone or Nr5a1 alone. Medaka cyp19a1a luciferase assay with fixed foxl2 (200 ng) or nr5a1 (100 ng) concentrations combined with variable concentrations of sdY (25–300 ng). Statistical significances of luciferase activity within treatments were tested using an unpaired two-tailed Student’s t test. The effect of foxl2 or nr5a1 alone compared with foxl2 or nr5a1 and variable concentrations of sdY was tested by a one-way ANOVA with post hoc Tukey tests. ***P < 0.001; **P < 0.01; *P < 0.1; ns, P > 0.05 (nonsignificant).