Fig. 2. Compound 3i inhibits expression of NRF2 target genes, reduces viability in lung cancer cells, and blocks migration. A549 or H460 cells were incubated for 24 h and (A) NRF2 protein levels or (B) mRNA expression in A549 cells were evaluated by western blotting or RT-PCR. NRF2 target genes were normalized to GAPDH and the DMSO vehicle control. Data are representative of 3 independent experiments. (C) Cells were treated for 72 h with various concentrations of 3i. Cell viability was measured using an MTT assay and values normalized as a percentage of vehicle (DMSO) control. n = 3 replicates. (D) Cells were synchronized in low serum media for 12 h, then treated for 24 h with inhibitors. Cell stage was analyzed by propidium iodide staining and flow cytometry, specifically univariate cell cycle analysis using a Watson pragmatic model with G0/1 and G2/M peak constraints (n = 2–3 independent experiments). Data are plotted as mean ± SE percentage of total cells. A549 cells were seeded in serum-free medium containing (E) DMSO vehicle, 10 μM of NRF2 pathway inhibitors MSU38225 or 3i; or (F) DMSO vehicle, and the indicated doses of 3i in the top chamber of transwells. Media containing compounds and 5% FBS as a chemoattractant was used in the bottom chamber and cells were allowed to migrate for 24 h, at which point transwell filters were fixed and unmigrated cells were removed. The remaining cells were stained with DAPI, and imaged (9 fields per transwell insert, representative image shown). Two transwell inserts were used for each condition, n = 2–3 experimental replicates. Data were analyzed by one-way or two-way ANOVA. * = p < 0.05; ** = p < 0.01; *** = p < 0.001; **** = p < 0.0001 vs. vehicle control. # = p < 0.05; ### = p < 0.001 vs. brusatol.