Figure 7: Increase in reactive oxygen species changes HSPC functions via TGF-β1 signalling.

(a) ROS detected by DCFDA staining in LSKCD48⁻ cell population from BM of 0T, 2T-WT and 2T-p190-B^−/− mice 5–7 weeks following transplant, analysed by flow cytometry. Data are presented as fold change in mean fluorescence intensity relative to 0T. (n=8–9 mice from three independent experiments (mean±s.e.m., two-tailed unpaired t-test). (b) Detection of aTGF-β by immunofluorescence staining: Effect of H₂O₂ and H₂O₂ +NAC treatments ex vivo for 12 h. Bar graph shows mean fluorescence intensity in arbitrary unit (mean±s.e.m.; 40–50 cells per experiment were analysed in each group, three independent experiments, two-tailed unpaired t-test). (c) 2T-WT mice were treated with NAC for 5–8 weeks following transplantation. 2T-WT mice not treated with NAC were used as controls. LSK-SLAM cells were immuno-stained for aTGF-β. Bar graph shows mean fluorescence intensity in arbitrary unit (mean±s.e.m.; 40–50 cells per experiment were analysed in each group, three independent experiments, two-tailed unpaired t-test). (d) Effect of H₂O₂ on 0T LSK-SLAM division output using the in vitro paired daughter cell assay. Single LSK-SLAM cells isolated from 0T-WT mice were treated with H₂O₂ or with H₂O₂+TGFBRI-Inh1 for the duration of one division. Daughter cells were analysed as in Fig. 1. Bar graph shows frequency of asymmetric and symmetric divisions (n=28 pairs per group, two independent experiments). P values were calculated by Fisher exact 2 × 2 contingency table by comparing per cent of symmetric and asymmetric divisions of H₂O₂ versus control, and of H₂O₂+TGFBRI-Inh versus H₂O₂ treatments.