Figure 4.

RhoA deficiency depletes HPCs but not HSCs in competitive setting. (A) CD45.2⁺ donor BM cells of the RhoA^fl/flMx-cre⁺ or Mx-cre⁻ genotype were cotransplanted into lethally irradiated CD45.1⁺ recipients together with equal number of CD45.1⁺ WT competitor cells. Three poly I:C injections were administrated 8 wk after the transplantation, and BM cells from the primary recipients were transplanted into secondary recipients 4 mo after the poly I:C injections. Data were analyzed 5 mo after secondary transplantation. Representative flow cytometry plots of CD45.2 expression within the LK, LSK, and LSKCD150⁺ population in the competitive transplant recipients are shown. (B–D) Relative chimerism of donor-derived LK (B), LSK (C), and LSKCD150⁺ (D) in BM of recipients. (E) DNA from sorted CD45.2⁺ LSK cells was used as PCR template to determine the deletion efficiency of RhoA. Data were analyzed 5 mo after secondary transplantation. Brightest band of ladder: 600 bp. (F) LSK cells from poly I:C–injected competitive transplantation recipients were transduced with RhoA-expressing or control virus and transplanted into secondary recipients. PB samples were examined 16 wk after transplantation. Percentage of donor-derived (CD45.2⁺) cells in gated GFP⁺ mature lineages of PB was determined by flow cytometry. Numbers of mice analyzed: six (A–D) and four (F) mice per group. The results from a representative experiment of three (A–D) or two (F) independent experiments are shown. Error bars indicate SEM. ***, P < 0.001.