Fig. 5

Loss of miR-31 results in compromised mammary tumor progression and reduces number of cancer stem cells. a Representative whole-mount staining of MMTV-PyVT (PyVT) and MMTV-PyVT/miR-31KO (PyVT/KO) mammary glands at 12 and 16 weeks of age. Quantification of PyVT tumor areas at 12 weeks (PyVT: n = 12; PyVT/KO: n = 9) and 16 weeks (PyVT: n = 12; PyVT/KO: n = 6) of age. Scale bar, 2.5 mm. b Gross images of mammary tumors from PyVT and PyVT/KO mice at 20 weeks of age. Quantification of mammary tumor volume from PyVT (n = 13) and PyVT/KO (n = 9) mice at 20 weeks of age. c H&E of PyVT and PyVT/KO tumors at 12 weeks of age. Higher magnification images indicated by dashed boxes shown in the lower panels. Arrowheads, nuclei with diverse shape. Scale bar, top panels, 50 μm; lower panels, 16 μm. d Immunohistochemistry for Ki67 and quantification of Ki67⁺ cells in PyVT and PyVT/KO mammary tumors at 12 weeks of age. Scale bar, 25 μm. e Immunostaining for Gata-3 and ERα in PyVT and PyVT/KO mammary tumors at 12 weeks of age. Scale bar, 25 μm. f Western blotting for Gata-3, ERα, K8 and K14 in PyVT and PyVT/KO tumors at 12 weeks of age. g Immunofluorescence for K14 and K8 at the leading edges of tumors and mammary ducts from PyVT and PyVT/KO mice. Scale bar, 25 μm. h qRT-PCR for miR-31 in CD24⁺CD90^- and CD24⁺CD90⁺ cells from PyVT tumors. n = 3 biological replicates. i Profiles of CD90-PE and CD24-PE-Cy7 in cell suspensions from PyVT and PyVT/KO tumors. Arrows, CD24⁺CD90⁺ cells. Quantification of CD24⁺CD90⁺ cells in PyVT (n = 4) and PyVT/KO (n = 4) tumors. j Limiting dilution transplanted assay showing tumor-initiating cell frequencies (with confidence intervals) χ²-values and associated probabilities. P = 0.0256. Data represented as mean ± S.E.M. for a, b; Data represented as mean ± S.D. for d, h, i. Sample size: PyVT (n = 3) and PyVT/KO (n = 3) for c–g. Two tailed unpaired t-test for d, h, i (*P < 0.05; **P < 0.01; ***P < 0.001)