Figure 3

Cyclin D1 is required for TGFβ-mediated cell migration. (A) SCP2 cells were transfected with Scr or cyclin D1 siRNAs and then treated with or without 5 ng/ml transforming growth factor beta(TGFβ) for 24 hours. Total cell lysates were analyzed for cyclin D1 and β-tubulin by Western blotting. (B) Representative images of phase contrast and wound mask of transfected SCP2 cells stimulated without or with TGFβ for 0 and 24 hours in scratch/wound healing assay. The initial wound mask (black) and wound closure (grey) were measured using the Essen Instruments Scratch Wound Module. (C) Relative wound width was analyzed by the IncuCyte™software (Essen Bioscience) and quantified for the indicated times (error bars indicate SEM; n = 3 independent experiments). (D) SCP2 cells were treated with either vehicle (dimethyl sulfoxide, DMSO) or mitomycin C (Mito C) in the presence or absence of TGFβ. SCP2 cell migration was quantified using wound closure area at 24 hours (error bars indicate SEM; n = 3 independent experiments). (E) Representative images of transfected SCP2 cells stimulated with or without TGFβ for 0 and 24 hours in Transwell cell migration assay. Cells were stained with 0.2% crystal violet. (F) Transfected and migrated SCP2 cells in Transwell migration assay were stained with DRAQ5 fluorescent dye and quantified using fluorescent density at 24 hours (error bars indicate SEM; n = 3 independent experiments). (G) SCP2 cells were transfected with empty vector, Flag-p21, and HA-cyclin D1 separately or in combination. TGFβ-mediated cell migration was assessed using the Transwell (top panel) and wound healing (bottom panel) assays. Migration of the cells was quantified using fluorescent density (Transwell assay) and wound closure area (wound healing assay) at 24 hours (Error bars indicate SEM; n = 3 independent experiments).