Constitutively active MEK1 (caMEK1) transiently rescues defects in Pak-deficient HSPC engraftment and proliferation. (A) 1.5 × 105 PID or MIEG3 (GFP+) cells co-transduced with caMEK, caAKT, or hCD4 empty vector control were co-transplanted with 5.0 × 105 CD45.1 whole bone marrow into lethally irradiated C57Bl/6J (CD45.2) recipient mice. Data represent mean ± SEM (N = 8-10 recipients per group). 2 independent experiments (**P < .01). Data shows the percentage of GFP cells in the peripheral blood at 3 weeks. (B-C) WT LSK were transduced with MIEG3 (black) or PID (gray) retrovirus and co-transduced with caMEK or hCD4 empty vector (hatched) control. At 36 hours posttransduction cells were stained using an anti-human CD4-PE Cy7 antibody, GFP+/PE Cy7+ double- positive LSK were then isolated by fluorescence-activated cell sorter. (B) Cell cycle analysis was performed 3 days postsort by culturing the isolated cells in the presence of BrdU for 1 hour and subsequent staining using an anti-BrdU antibody and 7AAD. Data represent the mean ± SEM;, percent of cells in the S phase of the cell cycle (N = 3 independent experiments), *P < .05. (C) Percentage of cells undergoing apoptosis was measured using Annexin V antibody and flow cytometry 3 days postsort. Data represent mean ± SEM (N = 3 independent experiments). *P < .05. ns, not significant.
