Figure 8. Tbx18 is a potent CArG box-dependent transcriptional repressor for smooth muscle differentiation.
(A-D) Transcriptional activity of CArG-box (open box) containing SMyA promoter-reporter constructs was measured in COS7 cells by luciferase assay with the indicated combination of expression plasmids encoding SRF, Tbx18, or empty vector. The results of at least three independent experiments performed in triplicate are presented. All values are normalized to cotransfected pSV40-Renilla luciferase activity. Values are presented as fold activation increases in luciferase activity ± standard error of the mean compared to the level of activity with empty expression vector alone. The promoter-reporter constructs used are shown schematically. *, p<0.05 compared with the SRF(-)/Tbx18(0) group. #, p<0.05 compared with the SRF(+)/Tbx18(0) group. (E-G) C3H10T1/2 cells were transfected with or without the V5-tagged Tbx18 expression vector (Tbx18OE and control, respectively) for 24 hours followed by TGF-β1 stimulation for 24 or 48 hours. Then cells were fixed and immunostained for V5 (green) plus SM22α (F, red) or SMγA (G, red). The percentage of SM22α+ or SMγA+ cells was quantified by scoring 3,000 cells in 10 non-overlapping fields each from 3 independent experiments (E) and shown as mean ± standard error of the mean. *, p<0.05 compared with non-transfected control.