Figure 5
The Wnt/beta-catenin pathway is indispensible in BMP2-induced C/EBPalpha promoter methylation. (a) Co-IP assay shows interaction between Dnmt 3a and Dnmt 3b, but not with GR in C3H10T1/2 cells after 21 days of treatment by BMP2 and 10–6 M Dex. (b) ChIP assay shows GR did not bind to C/EBPalpha promoter in C3H10T1/2 cells after 21 days of treatment as indicated. (c) The in vivo protein level of active beta-catenin in the distal femur area of Dex- or saline-treated mice. N=4. (d) The protein levels of active beta-catenin in C3H10T1/2 cells treated as indicated. (e) Relative expressions of Wnt target gene Axin2 and CyclinD1, as well as Wnt inhibitor DKK1 in C3H10T1/2 cells treated as indicated. (f) C3H10T1/2 cells were treated by BMP2 with or without 10–6 M IWR-1 for 7 days; the protein level of active beta-catenin and the mRNA level of Axin2 and CyclinD1 indicated the inhibition effect of IWR-1 on Wnt signaling. (g) The expression of osteoblast genes and adipocyte genes in C3H10T1/2 cells treated as indicated for 21 days. (h) In transdifferentiation assay, C3H10T1/2 cells were induced as indicated for 21 days before being switched to IFMD treatment. Adipocyte gene expression levels as well as transdifferentiated adipocytes were shown by qRT-PCR and oil red O staining, respectively. (i) DNA methylation status of C/EBPalpha promoter in C3H10T1/2 cells treated as indicated for 21 days (10 clones; Open circle: unmethylated CpG; full circle: methylated CpG, N=3). All mRNA expressions were normalized to beta-actin. Data are presented as mean±S.D. of three independent experiments. *P<0.05, **P<0.01, n.s. P>0.05, N=3. Densitometric analysis was shown as fold changes relative to beta-actin (western blot) or input (ChIP and Co-IP)
