Figure 4.
Retinoic acid upregulates RIG-I to stimulate Keap1-mediated proteasome degradation of NRF2. (A) Western blots for NRF2 in BMSCs, with or without ATRA treatment at the indicated time, after cycloheximide (CHX) treatment. β-Actin was used as a loading control. Quantified in supplemental Figure 4A. (B) Western blots for NRF2 in BMSCs, with or without treatment using ATRA and proteasome inhibitor MG132 or lysosome inhibitor chloroquine (CQ), as indicated. β-actin was used a loading control. Two biologically independent replicates have been presented; n = 6 biologically replicates. Quantified in Supplemental Figure 4B. (C) Western blots for Keap1 in BMSCs after ATRA treatment as indicated. β-actin was used as a loading control. Two replicates have been presented; n = 3–6 biologically replicates. Quantified in Supplemental Figure 4C. (D) Western blots for Keap1 in BMSCs, with or without ATRA treatment at the indicated time, after CHX treatment. β-Actin was used as a loading control; n = 3 biologically independent replicates. Quantified in supplemental Figure 4D. (E) Western blots for NRF2 and Keap1 in BMSCs with Keap1-knockdown and ATRA treatment as indicated. β-Actin was used as a loading control; n = 3 biologically independent replicates. Quantified in supplemental Figure 4E. (F) qPCR analysis of RIG-I, NRF2, and Keap1 in BMSCs after ATRA treatment as indicated. (G) Western blots for RIG-I, NRF2, and Keap1 in BMSCs after ATRA treatment as indicated. β-actin was used as a loading control. Two replicates have been presented; n = 4 biologically independent replicates. Quantified in supplemental Figure 4G. (H) Western blots for Flag-Trim25, HA-Keap1, and RIG-I in the input and immunoprecipitate with anti-Flag antibody from 293T cells. (I) Western blots for Trim25, Keap1, and RIG-I in the input and immunoprecipitate with anti-Trim25 antibody from BMSCs. (J) Western blots for HA-ubiquitin (Ub), HA-Trim25, Flag-Keap1, and RIG-I in the input and immunoprecipitate with anti-Flag antibody from 293T cells. *HA-Trim25. (K) Western blots for Ubiquitin (Ub), Keap1, and RIG-I in the input and immunoprecipitate with anti-Keap1 antibody from MG132-treated BMSCs obtained from Rig-I+/+ or Rig-I−/− mice. (L) Western blots for Ubiquitin (Ub), NRF2, and RIG-I in the input and immunoprecipitate with anti-NRF2 antibody from MG132-treated BMSCs obtained from Rig-I+/+ or Rig-I−/− mice. (M) Western blots for RIG-I, NRF2, and Keap1 in Rig-I+/+ or Rig-I−/− BMSCs after ATRA treatment as indicated. β-Actin was used as a loading control. Two replicates have been presented; n = 2-4 biologically independent replicates. Quantified in supplemental Figure 4H. (N) qPCR analysis of NRF2-induced genes in Rig-I+/+ or Rig-I−/− BMSCs after ATRA treatment as indicated. (O) Western blots for RIG-I, NRF2, Keap1, and Trim25 in Trim25-knockdown Rig-I+/+ or Rig-I−/− BMSCs, as indicated. Quantified in supplemental Figure 4I. (P) Illustration of the RIG-I-Trim25-Keap1 complex regulating NRF2 degradation. Error bars indicate mean ± SD. Repeated-measures 1-way (F) or 2-way (N) ANOVA followed by Dunnett’s test multiple comparisons. ‡P < .05; ‡‡P < .01; ‡‡‡P < .001; ‡‡‡‡P < .0001.

Retinoic acid upregulates RIG-I to stimulate Keap1-mediated proteasome degradation of NRF2. (A) Western blots for NRF2 in BMSCs, with or without ATRA treatment at the indicated time, after cycloheximide (CHX) treatment. β-Actin was used as a loading control. Quantified in supplemental Figure 4A. (B) Western blots for NRF2 in BMSCs, with or without treatment using ATRA and proteasome inhibitor MG132 or lysosome inhibitor chloroquine (CQ), as indicated. β-actin was used a loading control. Two biologically independent replicates have been presented; n = 6 biologically replicates. Quantified in Supplemental Figure 4B. (C) Western blots for Keap1 in BMSCs after ATRA treatment as indicated. β-actin was used as a loading control. Two replicates have been presented; n = 3–6 biologically replicates. Quantified in Supplemental Figure 4C. (D) Western blots for Keap1 in BMSCs, with or without ATRA treatment at the indicated time, after CHX treatment. β-Actin was used as a loading control; n = 3 biologically independent replicates. Quantified in supplemental Figure 4D. (E) Western blots for NRF2 and Keap1 in BMSCs with Keap1-knockdown and ATRA treatment as indicated. β-Actin was used as a loading control; n = 3 biologically independent replicates. Quantified in supplemental Figure 4E. (F) qPCR analysis of RIG-I, NRF2, and Keap1 in BMSCs after ATRA treatment as indicated. (G) Western blots for RIG-I, NRF2, and Keap1 in BMSCs after ATRA treatment as indicated. β-actin was used as a loading control. Two replicates have been presented; n = 4 biologically independent replicates. Quantified in supplemental Figure 4G. (H) Western blots for Flag-Trim25, HA-Keap1, and RIG-I in the input and immunoprecipitate with anti-Flag antibody from 293T cells. (I) Western blots for Trim25, Keap1, and RIG-I in the input and immunoprecipitate with anti-Trim25 antibody from BMSCs. (J) Western blots for HA-ubiquitin (Ub), HA-Trim25, Flag-Keap1, and RIG-I in the input and immunoprecipitate with anti-Flag antibody from 293T cells. *HA-Trim25. (K) Western blots for Ubiquitin (Ub), Keap1, and RIG-I in the input and immunoprecipitate with anti-Keap1 antibody from MG132-treated BMSCs obtained from Rig-I+/+ or Rig-I−/− mice. (L) Western blots for Ubiquitin (Ub), NRF2, and RIG-I in the input and immunoprecipitate with anti-NRF2 antibody from MG132-treated BMSCs obtained from Rig-I+/+ or Rig-I−/− mice. (M) Western blots for RIG-I, NRF2, and Keap1 in Rig-I+/+ or Rig-I−/− BMSCs after ATRA treatment as indicated. β-Actin was used as a loading control. Two replicates have been presented; n = 2-4 biologically independent replicates. Quantified in supplemental Figure 4H. (N) qPCR analysis of NRF2-induced genes in Rig-I+/+ or Rig-I−/− BMSCs after ATRA treatment as indicated. (O) Western blots for RIG-I, NRF2, Keap1, and Trim25 in Trim25-knockdown Rig-I+/+ or Rig-I−/− BMSCs, as indicated. Quantified in supplemental Figure 4I. (P) Illustration of the RIG-I-Trim25-Keap1 complex regulating NRF2 degradation. Error bars indicate mean ± SD. Repeated-measures 1-way (F) or 2-way (N) ANOVA followed by Dunnett’s test multiple comparisons. ‡P < .05; ‡‡P < .01; ‡‡‡P < .001; ‡‡‡‡P < .0001.

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