Caspase-3/7 activity assay

Caspase-3/7 activity was detected using a Caspase-Glo® 3/7 Assay kit (Promega). A total of 0.3 × 10⁶ cells of MCF-7 or MCF-7-AR6 were suspended in 1 mL of recommended media and transferred to a well of a 24-well ULA plate for 24 h at 37 °C in suspension. For transfected cells, at 48 h post-transfection, cells from each well of a 6-well tissue culture plate were trypsinized and combined with spent media. The cells were then centrifuged and resuspended in 2 mL of respective growth medium before being transferred into a well of a 6-well ULA plate for 24 h at 37 °C to induce anoikis. The caspase-3/7 activity in each sample was then measured according to the manufacturer’s protocol after 1 h incubation in the dark at 25 ˚C using GloMax®-Multi Jr Single Tube Multimode Reader (Promega).