Fig. 3.

aPKCζ and aPKCλ does not regulate HSC self-renewal, engraftment, and multilineage differentiation. (A) Experimental set up. The 2 × 10⁶ BM cells from CD45.2⁺ WT; Mx1-Cre or aPKCλ^f/f; Mx1-Cre or aPKCζ^−/−λ^f/f; Mx1-Cre mice were mixed with 2 × 10⁶ CD45.1⁺ B6.SJL^{Ptprca Pep3b/BoyJ} WT BM cells and competitively (1:1 ratio) transplanted into lethally irradiated primary mice. Serial transplantation of 1 × 10⁷ pooled BM cells from primary mice was performed into secondary and tertiary recipient mice. After BM engraftment (4 wk), aPKCλ deletion was induced by administration of pI-pC and CD45.2⁺ chimera was monitored in different time points. (B) Evolution (normalized to baseline) of CD45.2⁺ chimera in peripheral blood of primary recipient mice (n = 7–9 mice per group). (C) Evolution (normalized to baseline) of CD45.2⁺ chimera in peripheral blood of secondary recipient mice (n = 8 mice per group). (D) Representative FACS contour diagram showing CD45.2⁺CD11b⁺-myeloid and CD45.2⁺B220⁺-B-lymphoid cells during HSC engraftment. (E) Evolution (normalized to baseline) of myeloid-cell chimera (CD45.2⁺ cells gated on total CD11b⁺ graft) in the peripheral blood of primary recipient mice (n = 7–9 mice per group). (F) Evolution (normalized to baseline) of myeloid-cell chimera (CD45.2⁺ cells gated on total CD11b⁺ graft) in the peripheral blood of secondary recipient mice (n = 8 mice per group). (G) Evolution (normalized to baseline) of B-cell chimera (CD45.2⁺ cells gated on total B220⁺ graft) in the peripheral blood of primary recipient mice (n = 7–9 mice per group). (H) Evolution (normalized to baseline) of B-cell chimera (CD45.2⁺ cells gated on total B220⁺ graft) in the peripheral blood of secondary recipient mice (n = 8 mice per group). (B, C, E–H) Error bars represent SEM. (I) Normalized CD45.2⁺ chimera in peripheral blood of tertiary recipient mice at 8 wk after transplantation. n = 7–9 mice per group. Error bars represent SD.