Epcr expression supports HSC maintenance and supports engraftment of Mpl^−/−BM cells. (A) Expression of the anti-apoptotic genes Bcl-xL and Bcl-2 and (B) cell-cycle regulators Cdkn1a/p21 and Cdkn1c/p57 in LSK cells from mice transplanted with Epcr-transduced Mpl^−/− BM cells (Epcr, n = 3), GFP-transduced wt cells (positive control, n = 6) or GFP-transduced Mpl^−/− cells (negative control, n = 5). Shown is the expression relative to β-actin (2^{-CT genes}/2^{-CT actin}). LSK cells were sorted at >16 weeks after transplantation. For statistical analysis relative expression values were transformed using Y = Log(Y) (mean plus or minus SD; 2-tailed, unpaired Student t test with Welch correction [*P ≤ .05]). (C) Cell-cycle status of HSCs (LSK, CD34⁻, CD150⁺), determined by flow cytometric measurement of Ki-67 and 7-AAD, at >16 weeks after transplantation of GFP-transduced wt (positive control, n = 10), GFP-transduced Mpl^−/− (negative control n = 9), or Epcr-transduced Mpl^−/− BM cells (n = 6) (box-whisker plot; 2-tailed, unpaired Student t test with Welch correction [**P ≤ .01]). (D) LSK cell counts in the BM and spleen of Mpl^−/− mice transplanted with Epcr-transduced Mpl^−/− BM cells (n = 3), GFP-transduced Mpl^−/− (negative control, n = 5) or GFP-transduced wt lin⁻BM cells (positive control, n = 4) 1 week after transplantation, and 4 weeks after transplantation shown in panel E (separate transduction and transplantation experiment; Epcr, n = 4; negative control, n = 5; positive control, n = 4; mean plus or minus SD; 1-way ANOVA with Dunnett multiple comparisons test [#P ≤ .05]). (F) Cell-cycle status of HSCs (LSK, CD34⁻, CD150⁺) from mice transplanted with Epcr-transduced Mpl^−/− BM cells (n = 4) in comparison with the negative (n = 5) and positive control (n = 4) groups 4 weeks after transplantation (box-whisker plot; 2-way ANOVA with Tukey multiple comparisons test [##P = .009]).