Figure 5.

The Gabbr1^−/−mutant HSPC pool has reduced proliferative capacity. (A) Cell-cycle analysis. Left panel, Representative flow cytometry gates of cell-cycle analysis by Ki-67 and 7-AAD in LSK progenitors. Right panel, There are more cells in G0 phase in Gabbr1^−/− LSK progenitors when compared with Gabbr1^+/+ controls (n = 6 per group). (B-C) CFU assays were performed to determine proliferative and differentiative capacity of HSPCs. (B) Absolute numbers of progenitors were determined by plating equal numbers of BM (5 × 10⁴ cells) from WT or Gabbr1^−/− tibias. (C) The tritiated thymidine incorporation assay was used to determine the cell-cycling status of BM progenitors. (D) Stimulation with poly(I:C) (10 µg/g) was done by intraperitoneal injection into WT and Gabbr1^−/− mice (n = 4 per group). Forty-eight hours later, LSK progenitor number and cell cycle were analyzed. (E) BrdU incorporation assay. BrdU (50 mg/kg) was intraperitoneal injected into WT (n = 6) and Gabbr1^−/− mice (n = 5). Forty-eight hours later, percentages of BrdU⁺ cells in LSK and LSK CD48⁻ progenitor populations were analyzed. (F-I) In vitro B-cell differentiation assay. (F) Experimental design. Four thousand sorted LSK progenitors from WT and Gabbr1^−/− mice were seeded onto OP9 cells. After 1 week, cells were passaged onto fresh OP9 cells. At day 14, all cells were collected, counted, and stained with CD45 (G), CD19 (H; left panel), B220 (H; right panel), and CD11b (I) antibodies. The absolute numbers were presented as mean plus or minus SD (data were from 2 independent experiments with 4-5 replicates.). *P < .05; **P < .02; ***P < .001; ****P = 0 vs Gabbr1^+/+ controls. Analyses of panels A and D-I performed at P15; panels B and C performed at P13 and P15 (see also supplemental Figure 6).