Intrinsic role of Mysm1 in DC development. (A) CD45.2⁺ (2 × 10⁶) WT or Mysm1^−/− BM cells were transplanted into lethally irradiated CD45.1⁺ recipient mice at 1:1 ratios with CD45.1⁺ WT competitor cells (2 × 10⁶). CD45.2⁺ cell populations in recipients were determined by FACS at 3 weeks posttransplantation in BM, peripheral blood (PBL), and spleen (SP). The representative FACS data from 2 mice were shown. The experiments were repeated more than 3 times with similar results. (B) BM cells were harvested from the femurs and tibiae of Mysm1^−/− and WT littermates and 2 × 10⁶ cells/mL and were then plated in a 6-well plate. For Flt3L-induced DC differentiation, BM cells were cultured for 8 days without disruption in DC culture media supplemented with 100 ng/mL Flt3L. Cells were harvested and analyzed via FACS for pDCs (CD11c⁺B220⁺) and cDCs (CD11c⁺B220^-−). The experiments were repeated more than 3 times with similar results. (C) For GM-CSF-induced DC culture, BM cells were cultured for 5 days in DC culture media supplemented with 20 ng/mL GM-CSF and 20 ng/mL IL-4. Cells were harvested and analyzed via FACS for cDC (CD11c⁺CD11b⁺). (D) BM cells from Mysm1^−/− mice were transduced with either lentivirus-expressing mouse Mysm1 (LV-Mysm1) or control lentivirus (LV-GFP) for 8 hours. Cells were washed and cultured in Flt3L (100 ng/mL) supplemented media for 8 days and CD11c⁺ DC differentiation was analyzed by FACS. All the experiments were repeated more than 3 times with similar results. SSC, side scatter.