Figure 3.

The Lin^negcKit⁺CD71^lo/negpopulation of FL erythroid progenitors is the target of signaling through Vdr. (A) Representative flow cytometry plot showing the gating of E12.5 Lin^neg cKit⁺ FL cells based on CD71 expression. (B) Real-time RT-PCR analysis of Vdr RNA (10 ng) expression in ckit⁺CD71^lo/neg and ckit⁺ CD71^hi populations. Expression was normalized to Ubb (n = 3). (C) Distribution of colonies formed from ckit⁺CD71^lo/neg and ckit⁺ CD71^hi cells. Cells were sorted and cultured in methylcellulose (125 cells in 250 μL, 24-well dish) with or without calcitriol (n = 3). CD71^lo/neg, but not CD71^hi, cells responded to activation of Vdr (BFU-E + mCFU-E + calcitriol, 66% vs 49% − calcitriol). CD71^hi cells produced comparable numbers of CFU-E in the presence or absence of calcitriol (89% vs 85%, respectively). (D) Photograph of plates of colonies formed from ckit⁺CD71^lo/neg cells cultured in methylcellulose for 8 days. (E) Proliferation of ckit⁺CD71^lo/neg or CD71^hi cells cultured in MM with or without calcitriol. Stimulation of Vdr signaling resulted in a ∼2- to 3-fold increase in CD71^lo/neg cell proliferation (n = 5). Data were analyzed using an unpaired Student t test (B-C) or 2-way ANOVA (E; *P < .05, C; **P < .01, B,E; ***P < .001, E). Error bars, ± SEM.