Figure 6.
Inhibition of mTOR rescues HSC defects of Sel1L KO. (A-B) Chimerism maintenance analysis reveals rapamycin treatment rescued the loss of reconstitution potential of Sel1L KO HSCs. 5 × 105 CD45.2+ whole bone marrow cells from Mx1-cre+; Sel1Lfl/fl (fl/fl) or control mice (+/+) (without pIpC) were transplanted together with 5 × 105 CD45.1+ wild-type bone marrow cells into lethally irradiated CD45.1+ wild-type recipients. Transplants were injected with pIpC 6 weeks after transplantation and injected with rapamycin (4 μg/kg body weight) or vehicle daily starting 6 days before pIpC and continued throughout the experiment. The contribution of CD45.2 cells (A) in total CD45+, myeloid (Mac-1+), B (B220+), and T (CD3+) cells of peripheral blood, and (B) in HSCs and other hematopoietic populations in the bone marrow were analyzed in transplant recipients. Three independent experiments were pooled and n is described as numbers of replicates from each experiment, separated by the plus (+) sign. (C-D) Six- to 8-week-old Mx1-cre−; Rictorfl/fl or Mx1-cre−; Raptorfl/+; Rictor fl/fl (control) and Mx1-cre+; Raptorfl/+; Rictor fl/fl (DKO) mice were injected with pIpC every other day for a total of 6 doses. Two weeks after pIpC injection, frequency and proliferation (by Ki67) of HSPC were analyzed by FACS. (E-F) Six- to 8-week-old Mx1-cre−; Sel1L fl/fl; Rictorfl/fl or Mx1-cre−; Sel1L fl/fl; Raptorfl/+; Rictor fl/fl or Mx1-cre−; Sel1L fl/fl (control), Mx1-cre+; Sel1L fl/fl (Sel1L KO) and Mx1-cre+; Sel1L fl/fl; Raptorfl/+; Rictor fl/fl (triple knockout) mice were injected with pIpC every other day for a total of 6 doses. Two weeks after pIpC injection, frequency and proliferation (by Ki67) of HSPC were analyzed by FACS. Data represent mean ± SD except for panel A, where mean ± standard error of the mean were used. Two-sided Student t test was used for statistical analysis.