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. Author manuscript; available in PMC: 2013 Jul 3.
Published in final edited form as: Nat Cell Biol. 2013 Feb 24;15(3):309–316. doi: 10.1038/ncb2699

Figure 5. Nrf2 Mediates HSPC Functions via CXCR4.

Figure 5

(a) Representative flow cytometric analysis and (b) bar graphs showing the proportion of sorted WT and Nrf2−/− BM LSK cells expressing CXCR4; mean + SEM of 4 animals per strain; Δ MFI (Mean fluorescence intensity) = (MFI of WT LSKs) − (MFI of Nrf2−/− LSKs). (c) Representative flow cytometric analysis and (d) bar graphs showing the proportion of sorted WT and Nrf2−/− BM LT-HSCs expressing CXCR4; mean + SEM of 6 animals per strain. (e) Percentage of LSK cells that migrated toward unconditioned media with (+) or without (−) supplementation of CXCL12 at 6 hours of assays; mean + SEM of 6 observations per strain over 2 independent experiments. (f) Schematic diagram representing the regulatory region of Cxcr4 promoter. The positions of potential transcription factor binding sites are noted (bp). BS, putative Nrf2 Binding Site; TSS, Transcription Start Site. (g) HEK293T cells were transfected with Cxcr4 promoter-driven luciferase reporter plasmids (covering −0.5 kb from TSS), and luciferase activity was assessed 24 hours after transfection. Data represent the mean + SEM of 3 independent experiments. (h) Chromatin bound DNA from WT BM was immunoprecipitated with a Nrf2-specific antibody or IgG control. Quantitative PCR was performed using primers amplifying potential Nrf2 binding sites on Cxcr4 promoter. Data represent the mean of 2 independent experiments. (i - k) Lentiviral overexpression of CXCR4 in Nrf2−/− HSPCs. (i) Transduction efficiency of WT or Nrf2−/− LSKs with CXCR4-expressing vector (marked by expression of GFP) assessed 48 hours after transduction. (j) Representative flow cytometric analysis showing differentiation of WT or Nrf2−/− LSKs transduced to overexpress with CXCR4 then co-cultured with OP9-DL1 for 11 days, reproducible in 3 independent experiments. (k) Percentage of GFP-transduced or CXCR4-overexpressing WT and Nrf2−/− LSK cells migrated toward unconditioned media supplemented with CXCL12 at 6 hours of assays; mean + SEM of 5 independent experiments.