Genetic or pharmacological inhibition of Bcl-xL causes megakaryocyte apoptosis. (a) Caspase-3/7 activity in FLC-derived MGKs. Fetal livers were cultured in serum free media plus TPO for 3 d. Large MGKs were purified from a BSA-gradient and reseeded. 5 h later, caspase activity was measured using the Caspase-Glo assay system. Data represent fold increase compared with control ± SEM. n = 2 biological replicates for each genotype. (b) Viability of MGKs derived from fetal liver was measured 24 h after reseeding using the CellTiter-Glo assay system. Data represent mean ± SD. n = 3 independent experiments. Control was set as 100%. (c and d) MGKs derived as in a from fetal liver (FL) or BM (BM) were treated with ABT-737 (5 µM) or vehicle (DMSO) in serum-free media plus TPO. (c) Caspase-3/7 activity was measured after 5 h. Data represent fold increase compared with DMSO control. n = 6 independent experiments for FL; n = 3 for BM. (d) Viability after 24 h. n = 4 independent experiments. DMSO control was set as 100%. MGK were pooled from animals of same genotype. Mean ± SEM (e) Proplatelet formation by wild-type fetal liver–derived MGKs treated with ABT-737 or vehicle (DMSO). Data represent mean ± SEM. n = 6 technical replicates (except 5 µM, n = 2). Representative of three independent experiments. (f) Proplatelet formation by wild-type, FLC-derived MGKs treated with caspase inhibitors z-VAD.fmk or Q-VD-OPh or vehicle (DMSO). Data represent mean ± SEM, n = 2 technical replicates. Representative of three independent experiments. (g) Viability of MGKs incubated with z-VAD.fmk, Q-VD-OPh, or vehicle (DMSO). Cells were derived as in a from fetal liver and reseeded with the relevant agent. Viability was measured 24 h after reseeding using the CellTiter-Glo assay system. Untreated control was set as 100%. Data represent mean ± SD. n = 2 technical replicates in two independent experiments. *, P < 0.05; **, P < 0.005; ***, P < 0.0001.
