Murine rtpo

Mononuclear BM cells from WT or Fancc^−/− mice (CD45.2) were labeled with the lineage marker (Lin) biotinylated Abs mixture (BD Biosciences) including: anti- CD3ε (Clone 145-2C11), CD11b (Clone M1/70), CD45R/B220 (Clone RA3-6B2), Ly-76 (Clone Ter119), Ly6G, and Ly-6C (Clone RB6-8C5). Cells were then labeled with anti-Sca1-PeCy7 (D7) and anti-cKit-APC (2B8) (BD Biosciences). After washing, biotinylated Abs were revealed by Streptavidin Percp-Cy5.5 (BD Biosciences). Lin⁻Sca1⁺Ckit⁺ (LSK) cells were sorted using FACS Aria (BD Biosciences) from the CCHMC Flow Cytometry Core. Sorted LSKs (≥98%) were activated in StemSpan medium (Stemcell technologies) in presence of 25ng/ml murine rTpo (Preprotech) and 50ng/ml murine rScf (Preprotech). After 12h, LSKs were transduced by the 7TGC-eGFP Wnt reporter lentivirus (23 (link)) with 2 hits at MOI=5. Lentiviral particles were produced by the CCHMC Viral Vector core using 293T cells. At 72h, transduction efficiency was evaluated by mCherry detection by Flow cytometry (Transduction efficiency 70-80%). Five thousand LSK cells were transplanted into lethally irradiated Boy/J recipient mice (CD45.1). GFP expression of naïve splenic B cells was detected at 10 weeks post transplantation by Flow cytometry.

Briefly, BM Lin⁻ or LSK cells were maintained in StemSpan medium supplemented with 50 ng/ml murine rTpo (Preprotech, Rocky Hill, NJ), 50 ng/ml murine rSCF (Preprotech, Rocky Hill, NJ) and 1% BSA at 37 °C in normoxia (21% O₂, 5% CO₂). Cells with the indicated genotype were treated with increasing doses of the mitochondrial translation inhibitor Chloramphenicol (CAP; 0–60 μM) for 36 h followed by survival and Oxygen Consumption Rate (OCR) assays.