Figure 3.
Retinoic acid inhibits NRF2 to increase ROS levels and impair the function of BMSCs. Heatmap (A) and gene set enrichment analysis (GSEA) (B) of the indicated genes in control or ATRA-treated BMSCs in vitro. (C) Representative FACS plot (i) and quantification (ii) of cellular ROS levels in BMSCs, determined using H2DCFDA staining after ATRA treatment, as indicated; n = 4 biologically independent replicates. (D) ROS levels in BMSCs after ATRA and NAC treatment, as indicated; n = 3 biologically independent replicates. (E) Representative images (i) and quantification (ii) of CFU-F colonies formed by BMSCs treated with ATRA and NAC, as indicated; n = 5 biologically independent replicates. (F-G) Alizarin Red S staining (i) and quantification (ii) (F) and qPCR analysis of osteoblastic genes (G) in BMSCs, after induced osteoblastic differentiation. The osteogenic medium was administrated with vehicle or ATRA, as indicated; n = 3 biologically independent replicates. (H) Schematic of experimental design for NAC and ATRA treatment in vivo, as seen in panels I to N. (I-J) Representative FACS plot (i) and quantification (ii) of cellular ROS levels in BMSCs (I) and absolute numbers of BMSCs (J) in indicated mice; n = 5 mice per group. (K) Representative images (i) and quantification (ii) of CFU-F colonies formed by BMSCs from indicated mice; n = 3 mice per group. (L) qPCR analysis of HSC niche factors in sorted PDGFRα+ BMSCs from indicated mice; n = 4–6 biologically replicates. (M-N) The absolute numbers of recovered CD45.1+ donor-derived HSPCs (M) and cell cycle of CD45.1+ donor-derived HSCs (N) in recipient mice at 8 weeks after transplantation; n = 4-6 mice. (O-P) GSEA analysis of NRF2 pathways (O) and heatmap of NRF2-induced genes (P) in BMSCs after ATRA treatment. (Q) Western blots (i) and quantification (ii) for NRF2 protein levels in BMSCs after ATRA treatment. (R) Western blots for NRF2 in BMSCs with empty vector (EV) or NRF2 overexpression (OE) and vehicle control or ATRA treatment, as indicated. (S) ROS levels in BMSCs with EV or NRF2-OE and vehicle control or ATRA treatment as indicated; n = 3 biologically independent replicates. (T) qPCR analysis of HSC niche factors in BMSCs with EV or NRF2-OE and vehicle control or ATRA treatment, as indicated; n = 3 biologically independent replicates. (U) Chip-qPCR of Scf and Igf1 enrichment in BMSC anti-NRF2 precipitates as indicated. Error bars indicate mean ± SD. Black bars represent individual genes in rank order. NES, normalized enrichment score; FDR, false discovery rate. (B, O) Repeated-measures 1-way ANOVA (C-F, I-M, Q, and U) or 2-way (G, N, S, and T) ANOVA followed by Dunnett’s test for multiple comparisons, ‡P < .05; ‡‡P < .01; ‡‡‡P < .001; ‡‡‡‡P < .0001.

Retinoic acid inhibits NRF2 to increase ROS levels and impair the function of BMSCs. Heatmap (A) and gene set enrichment analysis (GSEA) (B) of the indicated genes in control or ATRA-treated BMSCs in vitro. (C) Representative FACS plot (i) and quantification (ii) of cellular ROS levels in BMSCs, determined using H2DCFDA staining after ATRA treatment, as indicated; n = 4 biologically independent replicates. (D) ROS levels in BMSCs after ATRA and NAC treatment, as indicated; n = 3 biologically independent replicates. (E) Representative images (i) and quantification (ii) of CFU-F colonies formed by BMSCs treated with ATRA and NAC, as indicated; n = 5 biologically independent replicates. (F-G) Alizarin Red S staining (i) and quantification (ii) (F) and qPCR analysis of osteoblastic genes (G) in BMSCs, after induced osteoblastic differentiation. The osteogenic medium was administrated with vehicle or ATRA, as indicated; n = 3 biologically independent replicates. (H) Schematic of experimental design for NAC and ATRA treatment in vivo, as seen in panels I to N. (I-J) Representative FACS plot (i) and quantification (ii) of cellular ROS levels in BMSCs (I) and absolute numbers of BMSCs (J) in indicated mice; n = 5 mice per group. (K) Representative images (i) and quantification (ii) of CFU-F colonies formed by BMSCs from indicated mice; n = 3 mice per group. (L) qPCR analysis of HSC niche factors in sorted PDGFRα+ BMSCs from indicated mice; n = 4–6 biologically replicates. (M-N) The absolute numbers of recovered CD45.1+ donor-derived HSPCs (M) and cell cycle of CD45.1+ donor-derived HSCs (N) in recipient mice at 8 weeks after transplantation; n = 4-6 mice. (O-P) GSEA analysis of NRF2 pathways (O) and heatmap of NRF2-induced genes (P) in BMSCs after ATRA treatment. (Q) Western blots (i) and quantification (ii) for NRF2 protein levels in BMSCs after ATRA treatment. (R) Western blots for NRF2 in BMSCs with empty vector (EV) or NRF2 overexpression (OE) and vehicle control or ATRA treatment, as indicated. (S) ROS levels in BMSCs with EV or NRF2-OE and vehicle control or ATRA treatment as indicated; n = 3 biologically independent replicates. (T) qPCR analysis of HSC niche factors in BMSCs with EV or NRF2-OE and vehicle control or ATRA treatment, as indicated; n = 3 biologically independent replicates. (U) Chip-qPCR of Scf and Igf1 enrichment in BMSC anti-NRF2 precipitates as indicated. Error bars indicate mean ± SD. Black bars represent individual genes in rank order. NES, normalized enrichment score; FDR, false discovery rate. (B, O) Repeated-measures 1-way ANOVA (C-F, I-M, Q, and U) or 2-way (G, N, S, and T) ANOVA followed by Dunnett’s test for multiple comparisons, ‡P < .05; ‡‡P < .01; ‡‡‡P < .001; ‡‡‡‡P < .0001.

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