Inhibition of Pak in HSPC results in impaired proliferation and increased apoptosis in vitro. WT LSK was transduced with PID (gray) or MIEG3 (black) retrovirus, and then GFP+ LSK were isolated by fluorescence-activated cell sorter. (A) Cell cycle analysis was performed by culturing the sorted cells in the presence of BrdU and subsequent staining using an anti-BrdU antibody and 7AAD. Data represent the mean ± SEM, percent of cells in S, G0/G1, or G2/M phase of cell cycle (N = 3 independent experiments). *P < .05; **P < .01. (B) The percent of cells undergoing apoptosis was measured using Annexin V/7AAD and flow cytometry. Data represent mean ± SEM (N = 3 independent experiments). *P < .05. ns, not significant. (C-D) Activity of signaling pathways were assessed using intracellular fluorescence-activated cell sorter and phospho-specific antibodies. (C) Representative flow cytometric analysis of levels of phospho-AKT, and ERK in MIEG3 or PID-transduced LSK, after serum starvation (solid line) or stimulation (dotted line) with cytokines for 5 minutes. Representative data from 1 of 3 independent experiments. (D) Composite data from MIEG3 (○) and PID (♦) transduced LSK cells. Data shows fold increase of phosphorylation for each group after stimulation, (MFI) ± SEM (N = 3 independent experiments). P < .05 for pAKT and pERK.