Figure 3.

Cited2 Maintains HSCs in a Cell-Autonomous Manner

Cited2^fl/flMx1-Cre and control mice were treated with pIpC as shown in Figure 1B.

(A) CFC assay performed on total BM cells from Cited2^Δ/Δ and control mice. The graph shows the mean number of CFC colonies ± SD counted on day 10 (n = 3 per group).

(B) CAFC assay. The graph shows the mean number of cobblestone areas ± SEM counted at week 5 (n = 3).

(C) Competitive repopulation assay. CD45.2⁺ BM cells from Cited2^Δ/Δ or control mice were mixed with CD45.1⁺ WT competitor BM cells and transplanted into irradiated CD45.1⁺ WT recipients. After 16 weeks, the contribution of CD45.2⁺ cells was analyzed. Data are mean percentage of CD45.2⁺ cells in PB of recipient mice ± SD (n = 6 per group).

(D) Frequencies of the BM LSK and Lin⁻Sca-1⁻c-Kit⁺ (LK) cells from Cited2^Δ/Δ, Cited2^+/Δ, and Cited2^fl/fl control mice. The data are representative of four independent experiments.

(E) BM cells from untreated Cited2^fl/flMx1-Cre and Cited2^fl/fl mice were mixed with CD45.1⁺ WT competitive BM and transplanted into irradiated recipients. Eight weeks after tansplantation, the mice were treated with five doses of pIpC. Five days after last pIpC administration, the percentage of test CD45.2⁺ cells was measured in BM LSK and LK compartments. Data are mean ± SD (n = 3). ^∗p < 0.001; ^∗∗p < 0.0002.

(F) LTC-IC assay. Human CD34⁺ CB cells transduced with shRNA and control lentiviruses were cocultured with MS5 stromal cells. After 5 weeks, medium was replaced with complete methylcellulose. The graph shows the mean percentage of GFP⁺ LTC-IC colonies in cultures ± SD (n = 2) scored 2 weeks after adding methylcellulose.