Fig. 5.

NSP14 inhibits SIRT1-mediated NRF2 signaling.A Real-time PCR analysis of NRF2 and HMOX1 mRNA expression. Huh7 cells were transfected with pooled siRNA against SIRT1 or PGC-1α for 48 h. Scramble siRNA was used as the control. Relative transcription levels of NRF2 and PGC-1α were quantified by real-time PCR and normalized to the level of GAPDH. B, C Real-time PCR analysis of HMOX1 mRNA expression mediated by different NRF2/HMOX1 agonists. Forty-eight hours post-transfection with siRNA against SIRT1 or PGC-1α, Huh7 cells were treated with hemin (10 μM), sulforaphane (10 μM), and CoPP (5 μM) for 6 h and then subjected to real-time PCR. D, E ARE-luciferase assays. HEK293T cells were transfected with Renilla luciferase control plasmid, firefly luciferase reporter plasmid ARE-Luc, and increasing amounts of SIRT1-expressing plasmid for 24 h and then treated with sulforaphane and tert-butylhydroquinone (tBHQ) for an additional 24 h. The protein samples were harvested for luciferase detection. F HEK293T cells were transfected with the Renilla luciferase control plasmid, firefly luciferase reporter plasmid ARE-Luc, HA-SIRT1, and Flag-NSP14 for 24 h following treatment with tBHQ for an additional 12 h. Then, cells were harvested for luciferase assay. Asterisks represent statistical significance based on two-tailed unpaired Student’s t test (*P  <  0.05, **P  <  0.01)