FIGURE 2.

PKCϵ mediates cyclin D1 hyperinduction in PKC agonist-treated IEC-18 cells. A, differential agonist-induced down-regulation of PKC isozymes in IEC-18 cells. i, cells were treated with PMA (P) or vehicle control (C) for the indicated times, and whole cell extracts were subjected to Western blot analysis of PKCα, PKCδ, PKCϵ, and cyclin D1 expression. Expression of eEF2α was evaluated as a loading control (L.C.). The individual lanes show analysis of the same whole cell extracts. Note the close correspondence between loss of PKCα expression and recovery of cyclin D1 levels (ii). B, overexpression of PKCϵ increases cyclin D1 levels in IEC-18 cells. Cells were transduced with adenovirus expressing LacZ, PKCα, PKCδ, or PKCϵ, as indicated, and expression of PKCα, PKCδ, PKCϵ, cyclin D1, and β-actin was determined by Western blotting. Data are from the same Western blot, with dashed lines indicating where lanes have been rearranged for clarity. C, effect of PKCϵ knockdown on PKC agonist-induced hyperinduction of cyclin D1. IEC-18 cells were transfected with non-silencing control siRNA (NS) or siRNA directed against rat PKCϵ (PKCϵ). After 48 h, cells were treated with PMA (P) or ethanol vehicle (E) for 6 h prior to analysis of cyclin D1 and PKCϵ expression by Western blotting. Fast Green staining of a representative area of the Western blot membrane is shown as a loading control. D, effects of overexpressing PKCα or PKCϵ on PKC agonist-induced changes in cyclin D1 expression. Cells transduced with adenoviral vectors expressing LacZ, PKCα (i), or PKCϵ (ii) were treated with PMA for the indicated times, and the expression of the indicated proteins was determined by Western blotting. Data are representative of at least three independent experiments.