HPP-4382 increases cellular glutathione levels: NHLF cells grown in 96-well Optilux plates were treated with compounds for 4 hours (BSO, 200 µM; Sulphorafane, 10 µM; CDDO-Me, 0.1 µM; HPP-1014, 10 µM; CoPP, 10 µM; HPP-4382 3 µM) and glutathione levels were determined using the GSH/GSSG-Glo Assay Kit (Promega).

<p>Positive values show pairs of means that are significantly different. All samples in duplicate, error bars represent standard deviation compared to DMSO. *, <i>p</i><0.05; **, <i>p</i><0.01, ***; <i>p</i><0.001.</p

HPP-4382 alters occupancies of Nrf2 and Bach1 on the HMOX1 E2 promoter.

<p>(A) NHLF Cells were treated with 0.1 µM CDDO-Me or 1 µM HPP-4382 for 6 hours after which they were crosslinked with 1% formaldehyde in media, washed, and collected to be processed for chromatin immunoprecipitation as described in <i>Materials and Methods</i>. Precleared nuclear lysates were incubated with antibodies against Nrf2 or Bach1. Immune complexes were than isolated with E.coli tRNA/Protein A agarose beads, and the obtained purified DNA with subjected to qPCR using primers for HMOX1 E2 promoter. *, <i>p</i><0.05 compared to the untreated sample of same antibody; n.s.  =  not significant. (B) NHLF cells were exposed to 20 nM Nrf2, Keap1, or control siRNA for 48 hours. Cells were lysed and separated via SDS-PAGE then Western blotted with antibodies against Nrf2, Keap1, or tubulin. (C) Cells transfected with either Nrf2 or control siRNA were subjected to chromatin immunoprecipitation after treatment with 1 µM HPP-4382 for 6 hours. Precleared nuclear lysates were probed with antibodies against Nrf2 or Bach1; a third set was not probed (mock). *, <i>p</i><0.01; **, <i>p</i><0.05. (D) Cells transfected with either Keap1 or control siRNA were subjected to chromatin immunoprecipitation after treatment with either 10 µM HPP-1014, 10 µM CoPP, or 1 µM HPP-4382 for 6 hours. Precleared nuclear lysates were probed with antibodies against Nrf2 or Bach1; a third set was not probed (mock). All samples were performed in triplicate, error bars represent standard deviation. *, <i>p</i><0.01; **, <i>p</i><0.05 compared to untreated siCtrl for same antibody probe. , <i>p</i><0.01; , <i>p</i><0.05 compared to untreated siKeap1 for same antibody probe.</p

Identification of molecules that induce HMOX1 expression.

<p>(A) Human lung fibroblast cells were plated in 384-well Optilux plates and screened with compound libraries at 15 µM for 18 hours. Cells were then fixed, permeabilized, and probed with anti-HMOX1 antibody. Fluorescence intensity of HMOX1 staining was quantified with a GE InCell imager. Control charts were prepared using the statistical software JMP. HMOX1-staining intensities greater than the upper confidence limit were deemed hits. (B) Representative images of cells expressing HMOX1 following compound treatment. NHLF cells were cultured in 96-well Optilux plates as described in <i>Materials and Methods</i>. Cells were treated with indicated compound at selected concentrations for 18 hours after which HMOX1 expression was determined by immunofluoresence and quantified on a GE InCell imager. (C) Potency of CoPP, HPP-1014, and HPP-4382 were determined in NHLF cells. Cells were treated for 18 hours, after which they were fixed, permeabilized, and HMOX1 expression determined via immunofluoresence captured on a GE InCell imager.</p

Heme binding motifs are required for activity of both CoPP and HPP-4382 on the HMOX E2 promoter.

<p>(A) Schematic representation of pFLAG-Bach1 (WT) and pFLAG-Bach1 (AP4-7) used in these experiments. (B) HepG2 cells were transfected with pGL-MARE-Luc plasmid DNA (containing the HMOX1 E2 promoter) plus a plasmid carrying either pFLAG-Bach1 (WT), pFLAG-Bach1 (AP4-7), or pFLAG-only for 24 hours. Cells were then transferred to 96-well plates and allowed to recover for 6 hours. After washing, Luciferase substrate was added for 30 minutes and fluorescence was measured on an Envision reader. (C, D) HepG2 cells were transfected and replated in 96-well plates as described in B, but treated with compounds at indicated concentrations (µM) overnight prior to determining luciferase activity. In (D), data is reported as fluorescence intensity fold over DMSO-treated cells in each set of transfection *, p<0.0001 compared to Bach1-WT expressing cells at same compound doses. Each sample was performed in quadruplicate, error bars represent standard deviation.</p