Fig. 8

Nox4 promotes survival of CAFs via an up regulation of Nrf2 pathway. (A) Western blot analysis showing expression levels of Nrf2 and KEAP1 in total lysates of CAFs versus RMFs. Relative signal intensity of Keap1 (normalized to the actin control) was shown in numerical numbers. (B) Total and phosphorylated (Ser349) levels of p62 in CAFs. (C) Levels of p62 in CAFs after 24 h of siNox4 transfection. (D) Starvation induced autophagy resulted in an increase in p62 accumulation in CAFs versus RMF. Fibroblasts were nutrient starved (HBSS buffer) for 6 h with or without treatment with the autophagy inhibitor, bafilomycin A1 (Baf; 100 nM). (E)Targeting Nrf2 impaired viability of CAFs compared to RMF. Confluent fibroblasts were treated with varying concentrations of brusatol and viability of cells were determined after 48 h of treatment. * represents p < 0.05 vs untreated CAFs, # represents p < 0.01 vs untreated CAFs. (F) Survival of CAFs after 48 h of siNox4 transfection in the presence or absence of DMF (30 uM). (G) Viability of CAFs after 48 h of sip62 transfection. # represents p < 0.01 vs untreated CAFs. (H) Oncomine microarray analysis of p62 mRNA expression in breast stroma vs normal stroma in the Finak et al. dataset. 1: Normal stroma, N = 6; 2: Tumor stroma, N = 53. (I) Inhibition of Nrf2 with brusatol induced caspase-3 activation in CAFs. T-Casp3 = total caspase-3; C-casp3 = cleaved caspase-3. (J) Bar graph—Relative mRNA expression of Birc5 in CAFs after 48 h of siNrf2 transfection. N = 3, * represents p < 0.05 vs siCon transfected CAFs. Right panel -Western blot analysis of Birc5 expression after 48 h of siNrf2 transfection. (K) Suppressing the expression of Keap1, a negative regulator of Nrf2, also resulted in an induction of Birc5 protein expression in CAFs. (L) Chemical induction of Nrf2 with DMF (ug) in RMF (left panel) or upstream overexpression of Nox4 with doxycycline (ng/mL) in the inducible RMF (iRMF.Nox4) (right panel) both increased the expression levels of Birc5. (M) Knocking down expression of Nox4 in CAFs inhibited expression of Birc5 in CAFs. Viability of CAFs when Birc5 was targeted either with 200 nM of YM155 treatment (N) or with siBirc5 transfection (O), for 48 h. N = 3, * represents p < 0.05 vs DMSO treated CAFs. (P) Collagen contraction activity of CAFs after 48 h of siBirc5 transfection. (Q) RT-PCR analysis of CAF markers, FAP and αSMA after 48 h of YM155 treatment. (R) Western blot analysis of Birc5 expression in primary breast CAFs vs. RMFs. All Western blots are representative of n = 3 separate experiments. Error bars are SD of n = 3