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. 2013 May 17;288(26):18911–18922. doi: 10.1074/jbc.M113.480996

FIGURE 3.

FIGURE 3.

Pin1 regulates cooperative action of Smad and Olig1. A, knockdown of Pin1 in NMuMG cells. Cells were transfected with control or Pin1 siRNA and harvested at the indicated times. Expression of Pin1 and α-tubulin (as a loading control) was analyzed by immunoblotting. In the following experiments, cells transfected for 48 h are used in each assay. B, knockdown of Pin1 inhibiting TGF-β-induced cell migration in a chamber migration assay. Cells were treated with TGF-β (1 ng/ml) or BMP-4 (10 ng/ml) for 12 h and subjected to chamber migration assays. Quantification is shown in the panel below. C, effects of Pin1 knockdown on Smad-Olig1 interaction. NMuMG cells were transfected with FLAG-tagged Olig1. Twenty-four h later, cells were stimulated with TGF-β (1 ng/ml) for 1 h and harvested. Cell lysates were subjected to immunoprecipitation (IP) with anti-Smad2/3 antibody, and co-precipitated Olig1 or Smad4 was visualized by immunoblotting (IB). D, effects of juglone, a Pin1 inhibitor, on Smad-Olig1 interaction. Cells were transfected with FLAG-tagged Olig1, treated with juglone (0.5–1.0 μm) for 24 h, stimulated with TGF-β (1 ng/ml) for 1 h, and harvested. Co-precipitation assay was performed as in C. E, effects of Pin1 knockdown on expression of TGF-β target genes. After knockdown of Pin1, cells were treated with TGF-β (1 ng/ml) for 1 h and harvested; mRNA levels of target genes were measured by quantitative real-time PCR. p values were determined by Student's t test. *, p < 0.05; **, p < 0.01. F, heat map of TGF-β-induced expression of target genes. Cells were transfected with control, Olig1, or Pin1 siRNA. After stimulation with TGF-β for 1 h, cells were harvested and subjected to DNA microarray analysis. Genes induced by TGF-β >1.6-fold in the siControl sample (151 genes) are shown. G, Venn diagram showing the overlap of Olig1-regulated genes and Pin1-regulated genes. Genes whose -fold induction by TGF-β were decreased by <0.7 after knockdown of Olig1 or Pin1 (siOlig1 or siPin1 fold/siControl fold < 0.7) are classified as Olig1-regulated genes or Pin1-regulated genes, respectively.