Figure 1.

PLK4 Inhibition Blocks Cell-Type Transition and αSMA Expression of Rat Adventitial Fibroblasts

Rat primary adventitial fibroblasts were cultured in the complete medium, starved in the basal medium overnight (see Methods), and pretreated for 30 min with vehicle (equal amount of DMSO) or the PLK4-selective inhibitor CenB at indicated concentrations, followed by stimulation with 60 ng/ml AA. Cells were harvested 24 h after stimulation (or as specifically indicated) for various assays. (A) Morphology. Cells were (or were not) stimulated by AA for 24 h without or with pretreatment (1 μmol/l CenB). Green fluorescent calcein was used to illuminate cell morphology. (B) Proliferation. CellTiter-Glo assay was performed after 72 h stimulation (by AA or solvent) without or with pretreatment with CenB at increasing concentrations. (C) Migration (scratch assay). Cells were (or not) stimulated by AA for 24 h without or with pretreatment (1 or 10 μmol/l CenB). Calcein was used to illuminate the cells. (D) Western blots of αSMA and vimentin. Protein band densitometry was normalized to loading control (β-actin) and then to the basal condition (DMSO, no AA), and finally quantified as fold change. Fold changes from at least 3 independent experiments were averaged, and mean ± SEM was calculated. One-way ANOVA/Bonferroni test: #p < 0.05; ##p < 0.01; ###p < 0.001. ∗p < 0.05; ∗∗∗p < 0.001 compared with vehicle control without AA (B) or with AA (C, D); ^ˆp < 0.05 between vehicle + AA and all other conditions in (B). αSMA = α-smooth muscle actin; AA = platelet-derived growth factor; ANOVA = analysis of variance; CenB = centrinone-B; DMSO = dimethylsulfoxide; PLK = Polo-like kinase.