Figure 5
At low inducer concentrations, C151 in KEAP1 is required for the ability of TBE-31 to activate expression of the HO-1 DsRed reporter in primary murine peritoneal macrophages. (A) NRF2 levels in total cell lysates of primary peritoneal macrophages isolated from WT or KEAP1C151S/C151S mice, that had been treated with vehicle (0.1% DMSO) or TBE-31 for 3 h. (B) Strategy for generating the HO-1DsRed/+ reporter mice. Deletion of the floxed region from the mouse Hmox1 flox allele by the Cre recombinase resulted in generation of the DsRed allele, in which the 3rd to 5th exons encoding an essential domain for the HO-1 activity are deleted, resulting in the expression of a DsRed fusion protein containing the N-terminal 48 amino acids of the HO-1 protein. (C) Representative fluorescence microscopy images of primary peritoneal macrophages isolated from WT-, KEAP1C151S/C151S- or NRF2−/−HO-1DsRed/+ reporter mice, that had been treated with vehicle (0.1% DMSO) or 10 nM TBE-31 for 24 h. Scale bar, 100 µm. (D) Quantification of the fluorescence of DsRed in primary peritoneal macrophages (n = 4) isolated from mice of the three genotypes following a 24 h-treatment with increasing concentrations of TBE-31. *p < 0.05.
