Figure 2. CKIγ2 inhibits Smad3-mediated transcription.

(a) Validation of CKIγ2 shRNAs. Flag-CKIγ2 (1µg) was co-transfected into 293T cells with the indicated pSuperRetro plasmids (pSR, 3 µg). pSR-GL2 contains the shRNA against luciferase and was used as a non-targeting control. Cells were lysed at 24 h post-transfection and total cell lysates were blotted for Flag-CKIγ2. γ-Tubulin was used as the loading control in this and most later experiments. (b) SBE-Lux luciferase assay in HepG2 cells. The luciferase reporter (0.5 µg) was co-transfected with the indicated pSuperRetro plasmids (3 µg) before TGF-β (100 pm) or vehicle was added. Data were presented as average ± s.d. (c and d). Similar luciferase assays in HepG2 cells using the MBE-Lux reporter (c) or the ARE-Luc reporter (d, together with an equal amount of FoxH1). Cells were co-transfected with the reporter construct (1 µg) and the indicated pSuperRetro plasmids (4 µg of pSR-GL2 or a combination of pSR-CKIγ2-R1 and -R2, 2 mg each). Twenty-four hours later, cells were treated with TGF-β (100 pm) or the TβRI inhibitor, SB431542 (10 µm), for another 16 h before analysis. (e) 3TP-Luc luciferase assays in HaCaT stable lines (see Materials and methods). Cells were transiently transfected with the reporter construct (1 µg) and treated with or without TGF-β (100 pm). Note that overexpressed CKIγ2 was transiently knocked down in the CKIγ2-WT cells (lane 3) by three consecutive transfections with pSuper-CKIγ2. CKIγ2 protein levels in each HaCaT line are shown (bottom). Actin was blotted to show equal loading of lysates. CKIγ2, casein kinase 1 gamma 2; shRNA, short hairpin-type RNA; TGF-β, transforming growth factor-beta; TβRI, TGF-β receptor I.