SP600125 inhibits proliferation of HCD57 cells. Proliferation was assessed by an MTT assay (A) or viable cell count (B) of HCD57 cells cultured in the presence of EPO alone (no EPO) or EPO + DMSO vehicle (EPO + vehicle) or SP600125 (SP) as indicated for up to 96 hours. (C) BrdU pulse-chase studies to measure cell-cycle progression. HCD57 cells were cultured for 48 hours in the presence of EPO alone (no addition) or in the presence of EPO and SP600125 inhibitor or DMSO vehicle as indicated. Following a 30-minute pulse with BrdU, the cells were washed and cultured for 6 hours. Values indicate the percentage of BrdU⁺ cells entering G₁. (D) Graphic representation of cell-cycle progression in the presence or absence of SP600125. Following treatment with or without SP600125, cells were pulsed with BrdU and then analyzed for BrdU and propidium iodide 6 and 9 hours following the pulse. The percentage of the BrdU⁺ cells entering G₁ (i), exiting S phase (ii), or entering G₂/M (iii) are plotted against time. Graphs represent triplicate samples and error bars represent standard deviation from the mean. (E) Apoptosis was assessed by flow cytometry of propidium iodide–stained cells of HCD57 cells cultured in the presence or absence of EPO or EPO and SP600125 or DMSO vehicle (Vehicle) for 24 hours (▥) , 48 hours (▪), 72 hours (□) or 96 hours (▧) .