Figure 2. Smad complex represses the REGγ–proteasome pathway through SBE.

(a) H1299 and HaCaT cells were transfected independently with REGγ reporter construct (2 μg), treated with different doses of TGF-β for 24 h before lysis and were analysed for luciferase activity. Data are representative of three technical repeats with mean±s.d. (two-tailed Student’s t-test, *P<0.05, **P<0.005). (b) H1299 cells were co-transfected with REGγ reporter construct (2 μg) in combination with either Smad2/4 (100 ng) or Smad3/4 (100 ng) expression plasmids for 24 h and then analysed for luciferase activity. Data are representative of three technical repeats with mean±s.d. (two-tailed Student’s t-test, *P<0.05). (c) H1299 were incubated in the absence or presence of 5 ng ml⁻¹ TGF-β for indicated time points. Total RNA was isolated and subjected to quantitative RT–PCR. Data are representative of three technical replicates with mean±s.d. (two-tailed Student’s t-test, *P<0.05, **P<0.005). (d) H1299 cells were transfected with siRNA directed against Smad3 (20 nM). After 48 h of transfection, cells were treated with 5 ng ml⁻¹ TGF-β for 12 h and semiquantitative RT–PCR was performed to analyse REGγ, Smad3 and p21 mRNA levels. (e) HaCaT, HepG2, MCF-7 and H1299 cells were treated with 5 ng ml⁻¹ TGF-β and analysed by western blotting. (f) HaCaT, HepG2 and MCF-7 cells were transfected independently with siRNA specific for Smad3 (20 nM, for 48 h) and total RNA was isolated. Error bars show the mean±s.d. from three technical replicates (two-tailed Student’s t-test, *P<0.05, **P<0.005). (g) Schematic representation of putative SBE boxes in the REGγ promoter. The black arrows indicate the functional SBE in the REGγ promoter. (h) EMSA assay was performed using purified glutathione S-transferase (GST)-Smad3 protein. Fifty nanograms of GST-Smad3 protein were incubated with ³²P-radiolabelled probe containing SBE box from the REGγ promoter. (i) H1299 cells were transfected with wild-type (2 μg) or mutated (2 μg) SBE REGγ luciferase reporter constructs. Cells were then left untreated or treated with 5 ng ml⁻¹ TGF-β for 24 h and luciferase activity was measured. Error bars show the mean±s.d. from three technical replicates. Data are representative of three independent experiments (two-tailed Student’s t-test, *P<0.05). (j) H1299 cells were treated with 5 ng ml⁻¹ TGF-β for indicated time and ChIP analyses were performed with indicated antibodies.