a: Scheme of the DMBA/TPA two-stage chemical carcinogenesis model; mice were treated with phenformin (Phen) or with the vehicle control starting from week 13. b: Effects of phenformin on tumor progression; average volumes of all tumors on each mouse were calculated; data shown are means ± SEM of all mice (n=10) in each group. c-j: H&E (c) and immunofluorescence analyses (e, g, i) of tumors collected at 25 wks. Staining of the differentiation markers CK1, CK10 and loricrin are shown in red. DAPI (blue) stain identifies nuclei. d: Quantification of highly differentiated (H), medium differentiated (M) and low differentiated (L) tumors based on analysis of H&E staining (c) of tumors in the control and phenformin-treated groups. f, h, j: Quantification of relative expression levels of CK1, CK10 and loricrin, respectively. c-j: Tumors from six mice in each group were used for the quantitative analysis (n=6). * P<0.05, **P<0.01 by two-way ANOVA in b,c, and by Student’s two tailed t-test for others. All scale bars = 100 μm.