Fig. 1: NIC exposure either through smoking in breast cancer patients or upon direct treatment in breast cancer cells results into increased EZH2 expression.

a Immunohistochemical stained breast cancer patient samples from smoking and non-smoking individuals showing the difference in EZH2 expression. Insets show 10 times digitally magnified pictures of images captured with ×40 objective. b Scatter plot shows the percentage of EZH2-positive cancer cells in both the groups. c Difference in the intensity score of EZH2 expression in two groups is depicted in the graph. d Graph represents the Q score for EZH2 expression in tissue sections of smoking and never-smoked breast cancer patients. e (i) Western blot shows upregulated EZH2 expression upon NIC treatment in breast cancer cell lines including normal breast epithelial MCF-10A cells. e (ii) Quantification of western blot was done using software ImageJ. f Graph displays the relative fold change of EZH2 expression at mRNA level between control and NIC-treated MDA-MB-231 cells. g Increased EZH2 promoter activity was observed in luciferase assay upon NIC treatment in cancer cells. h (i), i (i) Immunoblot showing the depleted EZH2 protein level in MDA-MB-231 breast cancer cells upon DZNepA treatment and after EZH2si transfection, respectively, which was recovered following NIC treatment. h (ii), i (ii) Graphs showing normalized EZH2 expression calculated with respect to α-tubulin. Graphs are plotted with SD, which is calculated from indicated number of samples used for the study. Two-tailed paired Student's t-test and one-way ANOVA was used for statistical analysis. *P < 0.05, **P < 0.005, ***P < 0.001. Scale bar 50 µm