Incucyte s3 imager system

Apoptotic levels were assessed by the IncuCyte S3 Imager system (Sartorious, USA). Cell death was been quantified by the caspase-3/7 reagent (Sartorious, USA #4440), which generates a green fluorescent signal upon activation of apoptotic pathways. Following manufacture's protocol, the IncuCyte Caspase-3/7 dye was diluted 1:1000 in complete differentiating media for a final assay concentration of 5 μM. The amount of green signal was quantified after treatment with either siRNA or OICR during 72 h with an acquisition of 36 pictures for each well of a 12-well plate every 3 h. Measurements and analyses were performed with the IncuCyte S3 software (Sartorius, USA) by averaging the technical replicates and normalizing to the fluorescence detected at T = 0.

Cells were cultured in DMEM/F-12 complete medium without phenol red (Thermo Fisher Scientific) and treated as indicated. Live-cell images were acquired using the incubator-based Incucyte S3 imager system (Sartorius) equipped with 10× and 20× objective lenses. If not otherwise indicated, images were acquired every 2 hours for 72 hours. Mitotic arrest duration and subsequent cell fate were judged by morphological hallmarks as previously described (44 (link)). Cell growth (confluency) was monitored and quantified using the Incucyte automated live-cell analysis system according to the manufacturer’s protocol.