FIGURE 2.

FHL2 cooperates with Arkadia in activation of Smad3/Smad4-dependent transcription. A, 293T cells were transfected with the TGF-β-responsive luciferase reporter (CAGA)₁₂-Luc (0.1 μg) together with Arkadia or C937A and increasing doses of FHL2 (0.1 μg and 0.3 μg). TK-Renilla was used as internal control. Cells were treated with SBI prior to stimulation with Activin A for 1 h. The basal activity of the (CAGA)₁₂-Luc reporter cotransfected with empty vector was arbitrarily set at 1, and data are presented as mean induction in luciferase activity ± S.D. from duplicate samples. The results shown are representative of those from more than four independent assays. Bottom: expression levels of transfected Flag-tagged Arkadia and FHL2 determined by Western blotting (WB). B, HepG2 cells were transfected with the reporter (CAGA)₁₂-Luc (0.1 μg) together with Arkadia and increasing doses of FHL2 (0.1 μg and 0.3 μg). Cells were treated with SBI prior to stimulation with TGF-β for 1 h. Bottom: expression levels of transfected Flag-tagged Arkadia and FHL2 determined by Western blotting. C, knockdown of FHL2 in HepG2 cells. HepG2 cells were transduced with either control (shCtl) or FHL2 shRNA (shFHL2) lentiviral vectors (Santa Cruz Biotechnology). FHL2 expression was analyzed by Western blotting. D, reporter assay in HepG2 cells transduced with control shRNA (lanes 1 and 2) or FHL2 shRNA lentiviral vector (lanes 3 and 4). RNAi: RNA interference. Ctl: control. Cells were treated with SBI prior to stimulation with TGF-β for 1 h. E and F, luciferase assay in 293T cells transfected with either Gadd45b (E) or PAI-1 (F) promoter reporters with different doses of FHL2 (0.1 μg and 0.3 μg). 44 h after transfection, cells were stimulated with Activin A for 3 h. G, 293T cells were transfected with luciferase reporter (CAGA)₁₂-Luc along with FHL2 and Arkadia subfragments as indicated. Cells were stimulated with Activin for 3 h.