Fig. 4

Expression of inducible nitric oxide synthase (iNOS) is increased in breast carcinoma and mouse mammary tumours in response to stress. a Oncomine Cancer Mircoarray databases were used to analyse expression of NOS2 in the The Cancer Genome Atlas (TGCA) Breast database (n = 137). Expression was compared between normal breast tissue (n = 61) and invasive breast carcinoma (n = 79). b MCF-7 cells were exposed to cortisol (1 μM) for 30 minutes and 24 h and mRNA was extracted. cDNA was synthesised and amplified in the presence of gene-specific primers for NOS2 and β-actin using qRT-PCR. Cycle threshold (Ct) values for NOS2 were normalised against β-actin and fold change was calculated using the delta-Ct method. Data are mean ± SEM and significance was determined using one-way analysis of variance (post hoc Tukey multiple comparisons). c The 4T1 mouse mammary gland cells were transplanted into the fourth mammary fat pad of female BALB/C mice and the animals were randomised into groups exposed to either acute restrain stress or no stress. Tumours were harvested, fixed in paraffin and sectioned subsequent to immunohistochemical detection (IHC) of iNOS. Labelling was scored (0–3) according to intensity; representative panels are shown. The Mann-Whitney test was used to ascertain statistical significance. *Significant increase: *p < 0.05, **p < 0.01, ***p < 0.001