Figure 5.
Rig-I deletion recovers the clonogenicity and osteogenesis capacities of BMSCs under ATRA treatment. (A) Representative FACS plot (i) and quantification (ii) of cellular ROS level in Rig-I+/+ and Rig-I−/− BMSCs, with or without ATRA treatment, as indicated; n = 3 biologically independent replicates. (B-C) The absolute numbers (B) and cell death (C) of BMSCs in Rig-I+/+ and Rig-I−/− mice treated with vehicle or ATRA, as indicated; n = 3–4 mice per group. (D) Western blots for RIG-I and NRF2 in NRF2-knockdown BMSCs from Rig-I+/+ or Rig-I−/− mice, with β-actin used as a loading control. (E) Representative images (i) and quantification (ii) of CFU-F colonies formed by NRF2-knockdown BMSCs from Rig-I+/+ or Rig-I−/− mice treated with vehicle or ATRA, as indicated; n = 3 biologically independent replicates. (F-G) Alizarin Red S staining (i) and quantification (ii) (F) and qPCR analysis of osteoblastic genes (G) in NRF2-knockdown Rig-I+/+ and Rig-I−/− BMSCs after induced osteoblastic differentiation as indicated. The osteogenic medium was administered with vehicle or ATRA as indicated; n = 3 biologically independent replicates. (H) Strategy outline for the role of RIG-I in osteogenesis ability of ATRA-regulated BMSCs. (I-J) Representative images (I) and quantitative measurements for micro-CT analysis (J) of femurs from control or ATRA-treated Rig-I+/+ or Rig-I−/− mice as indicated; n = 4 mice per group. (K-L) Representative image (K) and quantification (L) of OPN-expressing cells in distal femur metaphysis in control or ATRA-treated Rig-I+/+ or Rig-I−/− mice as indicated. Error bars indicated mean ± SD. Repeated-measures 2-way (A-C, E-G, J, and L) ANOVA followed by Dunnett’s test multiple comparisons. ‡P < .05; ‡‡P < .01; ‡‡‡P < .001; ‡‡‡‡P < .0001.

Rig-I deletion recovers the clonogenicity and osteogenesis capacities of BMSCs under ATRA treatment. (A) Representative FACS plot (i) and quantification (ii) of cellular ROS level in Rig-I+/+ and Rig-I−/− BMSCs, with or without ATRA treatment, as indicated; n = 3 biologically independent replicates. (B-C) The absolute numbers (B) and cell death (C) of BMSCs in Rig-I+/+ and Rig-I−/− mice treated with vehicle or ATRA, as indicated; n = 3–4 mice per group. (D) Western blots for RIG-I and NRF2 in NRF2-knockdown BMSCs from Rig-I+/+ or Rig-I−/− mice, with β-actin used as a loading control. (E) Representative images (i) and quantification (ii) of CFU-F colonies formed by NRF2-knockdown BMSCs from Rig-I+/+ or Rig-I−/− mice treated with vehicle or ATRA, as indicated; n = 3 biologically independent replicates. (F-G) Alizarin Red S staining (i) and quantification (ii) (F) and qPCR analysis of osteoblastic genes (G) in NRF2-knockdown Rig-I+/+ and Rig-I−/− BMSCs after induced osteoblastic differentiation as indicated. The osteogenic medium was administered with vehicle or ATRA as indicated; n = 3 biologically independent replicates. (H) Strategy outline for the role of RIG-I in osteogenesis ability of ATRA-regulated BMSCs. (I-J) Representative images (I) and quantitative measurements for micro-CT analysis (J) of femurs from control or ATRA-treated Rig-I+/+ or Rig-I−/− mice as indicated; n = 4 mice per group. (K-L) Representative image (K) and quantification (L) of OPN-expressing cells in distal femur metaphysis in control or ATRA-treated Rig-I+/+ or Rig-I−/− mice as indicated. Error bars indicated mean ± SD. Repeated-measures 2-way (A-C, E-G, J, and L) ANOVA followed by Dunnett’s test multiple comparisons. ‡P < .05; ‡‡P < .01; ‡‡‡P < .001; ‡‡‡‡P < .0001.

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