Genistein

The triazole compounds and derivatives were synthesized in the “Laboratório de Síntese Orgânica” (LSO) and physiochemically characterized as described previously [37 (link),38 (link)]. The remaining compounds were purchased from commercial sources at the analytical grade: VX-770 (#HY-13017), genistein (#HY-14596), forskolin (Fsk, #HY-15371), and CFTR channel inhibitor (CFTRinh-172, #16671) are from MedChemExpress (Monmouth Junction, NJ, USA); while VX-445 (#S8851) is from Selleckchem (Houston, TX, USA). All compounds were diluted in dimethyl sulfoxide (DMSO).

PPT, DPN, MPP, PHTPP, E₂, forskolin, genistein, rottlerin, wortmannin, GSK-7975A, ML-9, SKF-96365, CPA, and BAPTA-AM were purchased from MedChemExpress and dissolved in DMSO. CCh and indomethacin were purchased from Sigma-Aldrich. CCh was dissolved in ultrapure water. indomethacin was dissolved in anhydrous alcohol. All salts were supplied by Sangon Biotech and dissolved in ultrapure water.

Ovarioles were dissected from ovaries of 7-day-old adult females and desheathed in medium containing 60% Schneider’s Drosophila medium and 40% Basic Medium Eagle as previously described [58 (link)], followed by incubation with fresh medium containing 0.1 μM JH III (Santa Cruz) for 1 h. In pharmacological experiments with inhibitors, ovarioles were separately exposed to ACPD (0.1 μM), ML141 (10 μM, MedChemExpress), suramin (1 μM, Sigma-Aldrich), genistein (10 μM, MedChemExpress) and Su6668 (10 μM, MedChemExpress) for 30 min, followed by JH III (Santa Cruz) treatment for additional 1 h. DMSO was used as the control. After the incubation, CellMask Orange Plasma Membrane Stain (Invitrogen) was added and further incubated for 5 min. The images were captured with a ZEISS LSM710 laser confocal microscope and processed with Zen2012 software (Carl Zeiss). Area of follicle cell and patency were measured with Image-Pro Plus 6.0 (NIH). Patency index was calculated by comparing the area of patency surrounding a follicle cell to the area of this follicle cell.