Figure 2
Increased endogenous ROS production in BM of CXCR4−/− chimeras.
(a) Whole genome microarray analysis of CXCR4−/− and CXCR4+/+ LK and LSK cells. Venn diagrams show number of differentially expressed genes that are at least 1.5-fold changed. (b,c) GSEA of CXCR4−/− and CXCR4+/+ LSK cells showing significant enrichment of Hallmark Oxidative phosphorylation (b) and Hallmark DNA Repair (c) gene sets in CXCR4−/− cells. (d) Full gating strategy with actual staining plots for immunophenotypic analysis of HSCs from CXCR4+/+ and CXCR4−/− chimeras performed 10 weeks after FL cell transplantation. (e) Mean Fluorescence Intensity (MFI) of DCF on BM progenitor cells from CXCR4+/+ and CXCR4−/− chimeras according to the gating strategy described in (d). DCF staining showed increased intracellular ROS in CXCR4−/− LT-HSCs and HSPCs. Data are representative of 6 independent experiments. (f) DCF MFI of donor-derived LSK-SLAM (SLAM), LSK, LK and BM mononuclear (BM MNC) cells. Mean ± SEM from 6 mice per group, pooled from 2 experiments. (g) DCF MFI of LSK-SLAM cells from CXCR4+/+ and CXCR4−/− FL cells. Data from 3 independent experiments are shown. (h) Increased intracellular ROS in ten-week-old WT mice treated with CXCR4 antagonist TN140. Mean DCF MFI ± SEM from 6 mice per experiment in 2 independent experiments. (i) Increased phosphorylation of p38 MAPK in CXCR4−/− HSPCs. Mean phospho-p38 MFI ± SEM in donor-derived LSK-SLAM, LSK, LK and BM mononuclear (BM-MNC) fractions. Data from 8 mice per group over 2 independent experiments are shown. CXCR4+/+ and CXCR4−/− cells were obtained from chimeric mice 10 weeks after FL cell transplantation.
