Fig. 4. Loss of PKC reduces the ability of TGFβ to induce -SMA stress fibers, Smad3 phosphorylation, pro-fibrotic protein expression and matrix contraction in fibroblasts. (A) Immunofluorescence analysis. PKC^+/+ and PKC^-/- fibroblasts were cultured on fibronectin, serum starved for 18 hours and treated for an additional 24 hours with or without added TGFβ1 (4 ng/ml). Cells were fixed with paraformaldeyde and stained to detect -SMA. Cells were counterstained with DAPI to detect nuclei. (B) Western blot analysis and Smad phosphorylation. PKC^+/+ and PKC^-/- fibroblasts were cultured on fibronectin, serum starved for 18 hours and treated for an additional 0.5 hours with or without added TGFβ1 (4 ng/ml). Proteins were extracted, using 2% SDS, from cells. Equal amounts of protein (20 µg) were subjected to SDS/PAGE and western blot analyses with the antibodies indicated. (C) Western blot analysis and TGFβ-induced gene expression. PKC^+/+ and PKC^-/- fibroblasts were cultured on fibronectin, serum starved for 18 hours and treated for an additional 24 hours with or without added TGFβ1 (4 ng/ml). Proteins were extracted, using 2% SDS, from cells. Equal amounts of protein (20 µg) were subjected to SDS/PAGE and western blot analyses with the antibodies indicated. Note that basal protein expression is reduced in the absence of PKC. Moreover, expression of proteins in response to TGFβ is also impaired. (D) Floating collagen gel contraction assay. PKC^+/+ and PKC^-/- fibroblasts were seeded into a floating collagen gel matrix, and incubated for 24 hours in the presence and absence of added TGFβ1 as described in the Materials and Methods. The weight of the contracted collagen gel was then measured. Cells were assayed in triplicate; experiments were performed thrice. Values shown are average±s.d. ^*significantly different from control (P<0.05). (E) PKC^+/+ and PKC^-/- fibroblasts were seeded into a floating collagen gel matrix, in the presence or absence of the protein kinase C inhibitor calphostin C and analyzed as in C.