Mark microplate absorbance reader

The cell proliferation assay was conducted using the cell counting kit‐8 (HY‐K0301, MCE) following the manufacturer's instruction. Briefly, the cells were seeded into a 96‐well plate in triplicates at the density of 5000–8000 cells/100 µL. After 48 h, 10 µL dye solution was added and incubated at 37 °C for 3–4 h. The absorbance at 450 nm was measured using Mark microplate absorbance reader (Bio‐Rad). APC Annexin V apoptosis Detection kit (AO2001‐11P‐H, Tianjin Sungene Biotech Co., Ltd) was used for apoptosis assays following the manufacturer's instruction.

In order to corroborate the possible antiproliferative effect of the synthesized compounds, a MTT [3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay was carried out. Hence, 1 × 10⁴ cell/well) were cultured in 96-well plates with phenol red-free DMEM media as aforementioned. Synthesized compounds 6 and 37, and control drugs (Gossypol, a well-known Bcl-2 inhibitor and G-15 an antagonist of GPER), were added at increasing concentrations from 5 to 60 μM, subsequently, the cells were cultured for 72 h. The assay was performed by adding 100 μL of MTT (Sigma, St Louis, MO, USA) (0.25 mg/mL in clear media) to each well, and incubating for 1 h at 37 °C, 5% CO₂. After the time elapsed, the media was removed, and 200 μL of DMSO (reagent grade) were added to each well to dissolve the formazan crystals. Absorbance was measured at 540 nM in a Mark™ Microplate Absorbance Reader (Bio-Rad Laboratories, Hercules, CA, USA). Each data point was performed in sextuplicate in three different experiments, and the results were reported as the mean absorption ± SD.