ABT-263 reduces cell viability and potentiates CPT-induced apoptosis in hESCs, hiPSCs and NP.

<p>(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were treated with increasing concentrations of ABT-263 (0.1–1 μM) during 15h (top panel) or pre-treated with ABT-263 (0.1μM) for 1hour and then treated with CPT (1μM during 3h) (middle panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-263 (0.1μM) addition or 6 h after ABT-263 (0.1μM) and/or genotoxic removal. Results are presented as the percentage of the viability of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in quintuplicate. After 15 h of ABT-263 (0.1μM) addition or a withdrawal period of 3 h ABT-263 (0.1μM) and/or CPT-treated cells were harvested, and DNA oligomers were quantified by immunoassay (bottom panel). Results are presented as the percentage of DNA oligomers of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in triplicate. A paired Student's t test was used to compare ABT-263-treated samples with untreated ones (top panel) or CPT plus ABT-263 treated samples to CPT treated ones (middle and bottom panel)*P< 0.05, **P< 0.001. (b) Representative histograms of PI-stained ABT-263 (0.1μM) treated cells over a 20 h period or untreated unfixed cells. Percentage of PI positive cells (late apoptotic or necrotic) was determined by flow cytometric analysis (c) Caspase-9, caspase-3 activation and PARP cleavage in hESCs, hiPSCs and NP upon CPT (1μM for 3h), ABT-263 (0.1μM for 15 h) or ABT-263 (0.1μM) for 1 h prior and during CPT treatment were analyzed by Western blotting 3 h post genotoxic removal. GAPDH was used as loading control.