Fig. 6
Loss of miR-31 represses metastasis to the lung. a Quantification of metastasis-free survival rates of PyVT and PyVT/KO mice at 12 and 16 weeks of age. 12 wks: PyVT (n = 16), KO/PyVT (n = 9); 16 wks: PyVT (n = 20), KO/PyVT (n = 10). b Representative histology of lungs from PyVT and PyVT/KO mice at 12 and 16 weeks of age. Arrows point to metastatic foci in the lungs. Quantification of metastatic tumors in the lungs. 12 wks: PyVT (n = 16), KO/PyVT (n = 9); 16 wks: PyVT (n = 20), KO/PyVT (n = 10). Scale bar, 100 μm. c Immunofluorescence for Vimentin in PyVT and PyVT/KO tumors at 12 weeks of age. Scale bar, 50 μm. d Immunohistochemistry for Slug in PyVT and PyVT/KO tumors at 12 weeks of age. Scale bar, 25 μm. e qRT-PCR analysis for Twist1, Bmi1, Zeb1 and Fzd3 in PyVT and PyVT/KO tumors at 12 weeks of age. n = 3 biological replicates. f Immunofluorescence for P-cadherin in tumors (top panels) and normal mammary ducts (bottom panels) from PyVT and PyVT/KO mice at 12 weeks of age. Scale bar, 25 μm. g Immunofluorescence for β1-integrin (ITGB1) and IHC for α2-integrin (ITGA2) in PyVT and PyVT/KO tumors at 18 weeks of age. Scale bar, 25 μm. h Western blotting for ITGA2 and ITGB1 in PyVT and PyVT/KO tumors at 18 weeks of age. GAPDH was used as a loading control. i Morphology of PyVT and PyVT/KO tumor cells into three-dimensional matrigel culture at indicated time points. Arrows point to protrusions. j Immunofluorescence for Vimentin in BT549 breast cancer cells treated with Scramble RNA and miR-31 mimics. Scale bar, 50 μm. n = 3 technical replicates. Data represented as mean ± S.D. Sample size: PyVT (n = 3) and PyVT/KO (n = 3) for c–i. Two tailed unpaired t-test for e (*P < 0.05; **P < 0.01; ***P < 0.001)
