Assessing microRNA-induced Apoptosis

BC cell lines were transiently transfected with reagent only (mock), miR-control, miR-145-5p, miR- 145- 3p, siRNA-control, or si-UHRF1 at 10 nM in 6 well tissue culture plates, as described previously [14 (link), 17 (link)–19 (link)]. Cells were harvested by trypsinization 72 hours after transfection and washed in cold phosphate-buffered saline. For apoptosis assays, double staining with FITC-Annexin V and propidium iodide was carried out using a FITC Annexin V Apoptosis Detection Kit (BD Biosciences, Bedford, MA, USA) according to the manufacturer's recommendations and analysed within 1 hour by flow cytometry (CyAn ADP analyzer; Beckman Coulter, Brea, CA, USA). Cells were identified as viable cells, dead cells, early apoptotic cells, and apoptotic cells using Summit 4.3 software (Beckman Coulter), and the percentages of early apoptotic and apoptotic cells from each experiment were then compared. As a positive control, we used 2 μg/mL cycloheximide.