Figure 3: Loss of p190-B modulates HSPC activity via TGF-β signaling.

(a) Effect of TGFBRI inhibitor 1 on cell division kinetics of LSK-SLAM isolated from 2T WT mice. Single cells were treated with TGFBRI inhibitor 1 or DMSO ex vivo for 72 h to determine division kinetics as in Fig. 1 (n=100 cells per group in 2 independent experiments). (b) Effect of TGFBRI inhibitor 1 on cell division output using the in vitro paired daughter cell assay. Single LSK-SLAM cells isolated from 2T-WT mice were treated with TGFBRI inhibitor 1 (n=36 pairs) or TGFBRI inhibitor 2 (n=7 pairs) or DMSO (n=14 pairs) for the duration of one division. Daughter cells were separated and further cultured individually with serum and cytokines without inhibitors to assess multilineage differentiation potential of daughter cells. (c) Effect of rTGF-β1 on 0T-LSK-SLAM division output using the paired daughter cell assay as in b. Single LSK-SLAM cells isolated from 0T-WT mice were treated with rTGF-β1 (10 pg ml⁻¹ and 5 ng ml⁻¹ n=18 pairs) for one division; daughter cells were analysed as in b. Bar graphs in B&C show per cent of asymmetric and symmetric divisions in each group, from at least two independent experiments. P values were calculated by Fisher exact 2 × 2 contingency table by comparing per cent of symmetric and asymmetric divisions of each inhibitor relative to DMSO in b, and rTGF-β1 treatment relative to control in c. (d) Schema of experimental design. LSK-SLAM were isolated from 1T-WT mice, cultured with SCF+TPO with TGFBRI inhibitor 1 (10 μM) or DMSO for 48 h and transplanted into recipients with competitor cells without inhibitor. Dot plot shows PB analysis 4 months post-transplant; per cent donor-cell chimera is shown. Bar graph shows donor-cell derived relative lineage reconstitution in PB, 4 months post-transplant (mean±s.e.m.; n=8 in 2 independent experiments). P value was calculated using 2-tailed unpaired t test.