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. 2014 Mar 13;289(18):12680–12692. doi: 10.1074/jbc.M114.558981

FIGURE 6.

FIGURE 6.

Loss-of-function analysis of C18ORF1. A, decreased expression of C18ORF1 mRNA. The expressions of C18ORF1 and β-ACTIN mRNAs as a negative control are shown. RT(+), with reverse transcriptase; RT(−), without reverse transcriptase. Cont, control siRNA; C18, C18ORF1-specific siRNA. B, effect of C18ORF1 siRNA on TGF-β-induced luciferase reporter activity. C18ORF1 siRNA or control siRNA was incorporated into cells. Subsequently, the luciferase assay was performed using a (CAGA)12-luc reporter with or without TGF-β. All values represent mean ± S.D. Significantly different from control siRNA in the presence of TGF-β: ***, p < 0.001. C, prolongation of TGF-β-induced Smad2 phosphorylation by C18ORF1 siRNA. C18ORF1 or control siRNA was incorporated into cells. Next, the cells were divided into six dishes and then stimulated with TGF-β for the indicated times. The expressions of phosphorylated Smad2 (upper panel), Smad2 (middle panel), and β-actin (lower panel) were observed using each specific antibody. D, mouse C18orf1(mC18orf1) rescues suppression of TGF-β-mediated responses by human C18ORF1-specific siRNA. All values represent mean ± S.D. Significantly different from human C18ORF1-specific siRNA-transfected cells in the presence of TGF-β: *, p < 0.05. E, enhancement of TGF-β-induced p21 expression by C18ORF1 siRNA. C18ORF1 or control siRNA was transfected into cells. Next, the cells were separated into two dishes, and cells in one dish were stimulated with TGF-β for 4 h. Upper panel, expression of C18ORF1 by RT-PCR; 2nd panel, expressions of phosphorylated Smad2; 3rd panel, expression of p21; lower panel, expression of β-actin. F, enhancement of TGF-β-induced TMEPAI and JUNB expression by C18ORF1siRNA. C18ORF1 or control siRNA was transfected into cells. Next, the cells were separated into four dishes, and the cells in one dish were stimulated with TGF-β for the indicated times. The expression of each molecule was detected by RT-PCR. Upper panel, expression of C18ORF1; 2nd panel, expression of TMEPAI; 3rd panel, expression of JunB; lower panel, expression of β-actin. WB, Western blot.