Analysis of apoptotic protein expression in Mk cultures. (A) Kinetics of active (cleaved) caspase-3 (triangles) correlates strongly with apoptosis as measured by AnnexinV/PI method (diamond) in cytokine-cocktail-stimulated Mk culture as assayed by flow cytometry. Data is from one representative experiment (n = 3). (B) Cells from cytokine-cocktail-stimulated Mk cultures were co-stained with DAPI (blue), and antibodies against CD41a (red) and GADD45A (green) from day 0 to day 15. Images shown are representative fields (n ≥ 3 per slide). Results are from one representative experiment (n = 3). Image acquired with 20× objective. (C) MAP3K5 (ASK1) protein expression (diamonds) in cytokine-cocktail-stimulated Mk cultures as represented by geometric mean fluorescence intensity of anti-MAP3K5-antibody stained samples relative to cells stained with isotype-matched control antibodies (representative data from n = 2 experiments). Protein expression correlates with average MAP3K5 (ASK1) mRNA expression (squares) in CD41a+ cells as measured by microarray compared to average expression in cytokine-cocktail-stimulated Mk cultures from day 1 – 4 (n = 3). MAP3K5 and GADD45A exhibited similar protein expression patterns in cultures stimulated with Tpo alone (data not shown; n = 2).