Fig 2: MD2 inhibitors attenuate HOG-LDL-induced ER stress and ROS generation in MIO-M1 cells.a–c MIO-M1 cells were pretreated with or without MD2 inhibitors and exposed to HOG-LDL for 12 h, and ER stress was evaluated by determining the levels of GRP78 (a), ATF4 (b), and p-eIF2a (c). GAPDH was used as the loading control. d Cells were exposed to HOG-LDL for 3 h, and the subcellular localization of ATF6a (red) was determined by immunofluorescence staining. Cells were counterstained with DAPI (blue) (scale bar = 20 µm). e, f Cells were treated as described in (d) and stained with the ROS probe DCFH-DA. Fluorescence images (e) were captured (scale bar = 20 µm). The quantification of ROS levels was performed by flow cytometry (f). g, h The levels of NOX4 (g) and NOX4 subunit p22Phox (h) were measured in total lysates. Densitometric quantification was performed by normalizing the levels to GAPDH. Quantitative data are presented as the mean ± SEM, n = 3; #P < 0.05; ##P < 0.01; ###P < 0.001 compared to the DMSO control; *P < 0.05; **P < 0.01; ***P < 0.001 compared to HOG-LDL.

Fig 3: Real-time PCR analysis showing significantly higher Nox4 mRNA expression levels in breast CAFs, including the RMF-HGF, when compared to the normal mammary fibroblasts, RMF. (B) Western blot analysis of endogenous Nox4 expression in breast CAFs versus normal mammary fibroblasts. Representative of at least 3 analyses is shown. (C) AmplexRed assay indicating the levels of extracellular H2O2 in CAFs versus RMF after 4 h of incubation with the reagent. Fibroblasts were treated with DMSO control or GKT137831 (20 and 30 uM) for 24 h prior to the assay. Representative data from N = 3 independent experiments. Error bars are standard deviations of means. # represents P < 0.005 vs. RMF. * represents P < 0.001 in GKT treatment vs. DMSO in individual fibroblasts. $ represents P < 0.001 vs. DMSO of the corresponding CAFs. (D-F) Cellular glutathione analysis. RMF-HGF and CAFs show higher levels of oxidized glutathione, GSSG when compared to RMF (D), resulting in significantly lowered GSH:GSSH ratios (F). All bar graph data are mean ± SD of 3 separate samples. N = 3 independent experiments. * represents P < 0.05, # represents P < 0.005 versus RMF. (G) In situ detection of Nox4 mRNA transcription on a human breast cancer tissue microarray (TMA-BR8013) using RNAscope kit as described in Methods and Materials. Brown staining was mostly detected in the stroma region of tissues from breast carcinomas. Representative images are shown for each tumor grade. (H) Boxplot depicting the manual scores for NOX4 RNA (0–4) on the TMA from 90 BC cases and 10 normal breast tissues. ISH scores were generated at × 200 magnification and recorded using the RNAscope system scoring guidelines: 0 = no staining; 1 = 1 to 3 dots per stroma cell; 2 = 4 to 10 dots per stroma cell; 3 = more than 10 dots per stroma cell with less than 10% of stroma cells with dot clusters; and 4 = more than 10 dots per stroma cell with more than 10% of stroma cells with dot clusters. * represents p < 0.001 vs normal stroma. (I) Boxplot showing varying levels of Nox4 RNA expression (Average # of brown spots detected per cell in the stroma region) in different grades of breast cancer. * represents p < 0.005 vs normal stroma

Fig 4: A dual inhibitor of Nox1-Nox4 enzyme activity attenuates TGF-ß2 mediated F-actin stress fiber formation and aSMA expression. Serum-starved confluent cultures of primary TM cells were pretreated (2 hours) with SB-431542 (10 µM) or GKT137831 (20 µM) and incubated with vehicle (0.1% DMSO) or TGF-ß2 (5 ng/mL) for an additional 22 hours, as indicated. (A) Representative confocal fluorescent images of phalloidin stained F-actin stress fibers (green) or immunostained aSMA IV (red) proteins expressed in treated cells. Blue, DAPI-stained nuclei. Scale Bar, 100 µm. (B, C) Quantitative comparison of B phalloidin fluorescence or C aSMA immunofluorescence (N = 4–8 separate wells per condition, 2 fields per well, 15 cells per field). ***P < 0.0001, 1-way ANOVA with Tukey's post hoc analysis.

Fig 5: Effect of hypoxia on H2O2 production and its role in ANP secretion in beating rat atria.Data were expressed as the mean ± standard error of the mean (A, n = 5; B, n = 6). Cont, control; Hy, hypoxia; GLX, GLX351322, an antagonist of NOX4; NAC, N-Acetyl-D-cysteine, an antagonist of ROS. *p < 0.05 vs. control; #p < 0.05 vs. hypoxia.