Figure 4: NR4A1 directly induces AXIN2 expression.
From: Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-β signalling
(a) Immunoblot analysis of AXIN2 and NR4A1 in HaCaT, NMuMG and PC3 cells stably expressing NR4A1 or control cells. Actin was included as a loading control. (b) NR4A1+/+ and NR4A1−/− MEFs infected with or without Flag-NR4A1 lentivirus were lysed for immunoprecipitation (IP) with AXIN2 antibody followed by immunoblot. (c,d) qRT–PCR analysis of AXIN2 in HaCaT, PC3, NMuMG and HEK293T cells stably expressing NR4A1 (c) or depleted of NR4A1 (d). The mRNA level in the control was set to ‘1’ for each cell type (n=3). (e) qRT–PCR of AXIN2 in NR4A1+/+ and NR4A1−/− MEFs. Data are presented as mean±s.d. (n=3). (f) Sequences and locations of putative NGFI-B-response elements (NBRE) in human and mouse AXIN2 promoters and identified gluconeogenic genes24. (g,h) ChIP in HEK293T cells treated with or without 100 ng ml−1 TPA (g) or in NR4A1+/+ and NR4A1−/− MEFs treated with or without 2 μg ml−1 lipopolysaccharide for 4 h (h). Data represent mean±s.d. n=3. P<0.01 or P<0.001 by Student’s t-test. (i) Immunoblot of NR4A1 and AXIN2 in HEK293T cells with NR4A1 or AXIN2 depletion by lentiviral infection and induced with 100 ng ml−1 12-O-tetradecanoylphorbol-13-acetate (TPA) for 4 h. Co., non-targeting shRNA. Actin was included as a loading control. (j) PC3 cells infected with empty vector, NR4A1, control shRNA or AXIN2 shRNA as indicated were treated with TGF-β (5 ng ml−1) for the indicated time and then lysed for immunoblot analysis.
