Fig. 8
Identification of miR-31 direct targets. a, b qRT-PCR analysis for Axin1, Dkk1, Gsk3β, Smad3 and Smad4 in Control (n = 3) and miR-31 KO (n = 3) a, as well as Control (n = 3) and DTG (n = 3) b mammary epithelium. c Western blotting for Dkk1, Axin1, Gsk3β and Smad4 in Control (n = 3) and miR-31 KO (n = 3), as well as Control (n = 3) and DTG (n = 3) mammary epithelium. β-Tubulin was used as a loading control. d Ratio of luciferase activity of miR-31 mimics vs. scrambled RNA in wild type (WT) and mutant (Mut) 3’-UTR constructs based on 3 independent experiments. e RNA crosslinking, immunoprecipitation, and qRT-PCR (CLIP-PCR) assay for Dkk1, Axin1, Gsk3β, Smad3 and Smad4 upon Ago2 antibody immunoprecipitates in response to miR-31 inhibitor and scramble RNA (NC). IgG was used as a negative control. f qRT-PCR analysis for Smad3, Smad4, Axin1, Dkk1 and Gsk3β in PyVT (n = 3) and PyVT/KO (n = 3) tumors. g Western blotting for Dkk1, Axin1, Gsk3β, Smad4 and p-Smad2/3 in PyVT (n = 3) and PyVT/KO (n = 3) tumors at 12 weeks of age. β-Tubulin was used as a loading control. h Immunofluoresence for Dkk1 in PyVT (n = 3) and PyVT/KO (n = 3) tumors at 12 weeks of age. Scale bar, 50 μm. i Flow cytometry profiles of CD24-FITC and CD29-PE in cell suspensions of mammary epithelium from K5-rtTA (n = 3), DTG (n = 3), Dkk1 (n = 3) and DTG/Dkk1 (n = 3) mice. Arrows indicated CD24+CD29high cell population. n = 3 biological replicates. Data represented as mean ± S.D. n = 3. Two tailed unpaired t-test for a, b, d, f (*P < 0.05; **P < 0.01; ***P < 0.001)
