Fig 2: HDAC3 inhibition activates Nrf2 signaling by decreasing Keap1–Nrf2 interaction. a HUVECs were cultured in different condition, and the cell lysates were subjected to immunoprecipitation with Nrf2 antibody, followed by immunoblotting with indicated antibody. b and c The quantitative analysis of (a). d The cell lysates of HUVECs were subjected to immunoprecipitation with Nrf2 antibody, followed by immunoblotting with indicated antibody. e and f The quantitative analysis of (d). g Nrf2 nuclear translocation was determined in fixed cells by immunofluorescent staining in HUVECs. The red area represented Nrf2 and the nucleus was blue. Scale bars: 15 µm. The data are represented as the means ± SEM (n = 4). Significance: *P < 0.05 vs. MAN in scrambled HUVECs, #P < 0.05 vs. HG-PA in scrambled HUVECs, & P < 0.05 vs. HG-PA with si-HDAC3. All above results in graphs were normalized to first group

Fig 3: Treatment with Trastuzumab and/or Pertuzumab causes cytotoxicity and modulates expression of pHER2, pAKT, NRF2 and KEAP1 in ovarian cancer cell lines(A) Effect of antibodies on cell number. Cells were treated with Pertuzumab (PR), Trastuzumab (TR) or combination (COMB) of antibodies in the presence of 1nM Heregulin. Antibodies were used at 20µg/mL. After treatment, cell number was assessed by use of the MTT assay as described in Materials and Methods. Values are means with ± S.D of triplicates and expressed as fold decrease to HRG alone. Statistical significance was determined between untreated and treated groups by ONE WAY ANOVA followed by Tukey's post hoc test. Asterisks indicate significance of difference between HRG and individual treatment group whereas “#” indicates significance of difference between combination and Pertuzumab (red sign) or combination and Trastuzumab (blue sign) according to the scale * P <0.05, **P <0.01, ***P <0.001. (B) Western blot analysis showing levels of phospho HER2 T877 and phospho AKT S487 and (C) NRF2 and KEAP1 in response to treatments with 20µg/mL of Pertuzumab (Pr), Trastuzumab (Tr) and their combination (Comb) in PEO4, OVCAR4 and SKOV3 cells. Exponentially growing cells were treated with media containing 1nM Heregulin alone, or with co-treatment of Pertuzumab, Trastuzumab or their combination for 96h before being analyzed as described in Materials and Methods. The bar charts show phospho HER2 or NRF2 (red bars) and phospho AKT levels or KEAP1 (blue bars) following quantification of the immunoblot signal intensities obtained and normalized to HRG treatment group and expressed as fold change. The signal intensities of bands were quantified through integrated optical densitometry measurement using Gelpro software (Version 3.1, Media Cybernetics).

Fig 4: c-Met activation induces the Nrf2 pathway; and silencing of Nrf2 attenuates the cytoprotective function of c-Met against sorafenib-mediated ROS generation and apoptosis of renal cancer cells. a, b 786-O cells were incubated using combinations of HGF (50 ng/ml), sorafenib (10 µM), or only vehicle for 12–24 h. a Following isolation of nuclear and cytoplasmic fractions, Western blot was performed to check the level of Nrf2. Purities of nuclear and cytoplasmic fractions were examined through the expression of Sp1 and GAPDH respectively. b Cell lysates were immunoprecipitated (IP) with anti-Nrf2 antibody. The expression of Keap1 and Nrf2 in the immunoprecipitates were analyzed by Western blot. c–e After transfection with either Nrf2 siRNA (50 nM) or control siRNA for 48–72 h, 786-O cells were subjected to treatment (as in a) with combinations of HGF, sorafenib or vehicle for 24 h. c Cells were stained by using oxidative stress detection reagent and examined for endogenous reactive oxygen species (ROS) through flow cytometry. d The apoptotic cell death was checked through annexin V (APC) and propidium iodide stainings. The lysate of the transfected cells was analyzed for the expression of Nrf2 and ß-Actin by Western blot (right panel). e The cellular lysates were utilized for checking the levels of Bcl-2, Bcl-xL, cleaved Caspase-3, and ß-Actin through Western blot. a–e Results are representative of three different experiments

Fig 5: RNA sequencing reveals NRF2-activating mutation. (a) Schematic showing workflow for identifying an NRF2-activation gene signature. (b) Machine Learning Scores for sequential addition of genes identified 28 genes as the highest-scoring geneset. (c) Hierarchical clustering analysis of RNA sequencing data of all lung tumor (LUSC and LUAD) cases using Ward’s minimum variance method with the 28 gene signature. G1 was designated as the normal tissue cases, G2 as the NRF2-active group, and G3 as the NRF2-inactive group. Individual cases designated as normal, KEAP1-mutant, or NRF2 mutant are indicated with vertical lines below their clustered position. KEAP1 and NRF2 mutants were enriched in G2 relative to G3 (p < 0.05). R34 mutants were enriched in G2 relative to G3 (p < 0.05). (d) Heatmap showing coordinated increased expression of canonical NRF2 target genes in the G2 lung tumor cases.