Figure 4

SIRT6 interacts with NRF2 and positively regulates NRF2-ARE pathway. (A) Transcriptional activity of NRF2 in WT and SIRT6-deficient hMSCs was measured by ARE-driven luciferase reporter assay. WT and SIRT6-deficient hMSCs were transfected with pcDNA3.1 (vector) or pcDNA3.1-NRF2 (NRF2), together with ARE-luciferase and Renilla plasmids. Data were presented as mean ± SEM, n = 3, *P < 0.05. (B) Plasmid expressing GFP, SIRT6 (WT), or SIRT6 (HY) was transfected into hMSCs, together with NRF2 or vector, and then NRF2 activity was measured using ARE-driven luciferase reporter. Data were presented as mean ± SEM, n = 3, NS, not significant, *P < 0.05. (C) Effect of SIRT6 overexpression on activation of NRF2 target genes in primary hMSCs. hMSCs were transduced with luciferase (control), SIRT6 (WT), SIRT6 (HY), or NRF2, and then the HO-1 and AKR1C1 transcripts were determined by RT-qPCR. Data were presented as mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001. (D) WT and SIRT6-deficient hMSCs were transfected with a (UAS)₅-TATA-luciferase plasmid together with GAL4 or GAL4-NRF2, and then the NRF2 transactivity was measured. Data were presented as mean ± SEM, n = 3, NS, not significant, **P < 0.01. (E) Luciferase analysis of NRF2 transactivity in WT and SIRT6^−/− hMSCs in the presence of overexpressed GFP, SIRT6 (WT), or SIRT6 (HY). Data were presented as mean ± SEM, n =3, *P < 0.05, **P < 0.01. (F) ChIP-qPCR analysis of (UAS)₅-associated SIRT6 in hMSCs co-expressing (UAS)₅-TATA-luciferase, GAL4-NRF2, and Flag-SIRT6 using an anti-Flag antibody. Data were presented as mean ± SEM, n = 3, **P < 0.01. (G) Co-immunoprecipitation (Co-IP) showing that SIRT6 and NRF2 formed a protein complex. Exogenous (upper and middle panels) and endogenous (lower panel) Co-IPs were performed with the indicated antibodies. (H) GST-NRF2 or GST protein expressed from E. coli was incubated with Flag-SIRT6 expressed from HEK293T cells. The GST pull-down assay indicated an in vitro interaction between NRF2 and SIRT6.