Fig. 3

Effects of 5-methoxyflavone on the activation of Nrf2/HO-1 pathway in LPS-stimulated BEAS-2B cells. (A) Expression of Nrf2 pathway related proteins (Nrf2, HO-1) was detected by western blotting in LPS-stimulated BEAS-2B cells treated with 5-methoxyflavone for 12, 24 and 48 h. (B) Immunofluorescence analysis of 5-methoxyflavone-mediated Nrf2 nuclear translocation in LPS-stimulated BEAS-2B cells. (C) 5-Methoxyflavone activated the transcriptional activity of Nrf2 in LPS-stimulated BEAS-2B cells. BEAS-2B cells transfected with the pARE-Luc plasmid were treated with the indicated concentration of 5-methoxyflavone for 24 h, and then lysed for firefly and renilla luciferase activity measurement. Data was expressed as the ratio of firefly to renilla luciferase activity (Fluc/Rluc). (D) Expression of Nrf2, NOX4, TLR4, P-P65 and P-P38 was detected by western blotting in LPS-stimulated BEAS-2B cells treated with 5-methoxyflavone alone or in combination with brusatol treatment for 24 h. The band intensity of targeted proteins was determined by ImageJ software and was normalized to the intensity of GAPDH. ^##P < 0.01 versus the control group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus the LPS group; ^δP < 0.05, ^δδP < 0.01 versus the 5-methoxyflavone treatment alone