FIG 1.

TLR signaling activates the Nrf2 pathway. (A) RAW cells were treated with increasing doses of LPS for 16 h. HO-1 was assayed by Western blotting. (B) RAW cells were treated with 1 μg/ml of LPS for various times. HO-1 and GST were assayed by Western blotting. (C) RAW cells were treated with various doses of PGN for 16 h. HO-1 was assayed by Western blotting. (D) RAW cells were treated with various doses of poly(I·C) for 16 h. HO-1 was assayed by Western blotting. (E) WT mice were treated with LPS (10 μg/kg of body weight) by intraperitoneal injection for 24 h. Kidneys from two mice in each group were removed, and HO-1 was assayed by Western blotting. Con., control mice. (F and G) Time-dependent (F) and dose-dependent (G) study of Nrf2 protein levels. RAW cells were treated with 1 μg/ml LPS for different times or with different doses of LPS for 16 h, and the Nrf2 protein level was assayed by Western blotting. (H) RAW cells were treated with 0.1 and 1 μg/ml LPS for 3 h. Nuclear Nrf2 (N. Nfr2) was assayed by Western blotting. (I) Luciferase assay. HEK293 cells were transfected with the HO-1 promoter reporter plasmid pHO-1p/Luc, followed by treatment with increasing doses of LPS for 16 h. Cell lysates were assayed for luciferase activity. The experiments were repeated at least three times, and representative results are shown. *, P < 0.05 compared to the results for the control. (J) Cell viability assay. RAW cells were treated with various doses of LPS for 24 h. A cell counting kit (CCK-8) was used to measure the living cells. The assay was performed with cells in triplicate.