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Fig. 4 | Breast Cancer Research

Fig. 4

From: Upregulation of Nox4 induces a pro-survival Nrf2 response in cancer-associated fibroblasts that promotes tumorigenesis and metastasis, in part via Birc5 induction

Fig. 4

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

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