C2 ceramide reduces the ability of TGFβ to induce CCN2 protein in fibroblasts. a Fibroblasts were incubated for 24 h in DMEM/0.5% FBS. Cells were then incubated without or with C2 ceramide (cer, 10 μΜ, 45 min) or dihydroC2 ceramide (dihcer, 10 μΜ, 45 min) prior to incubation in the presence or absence of TGFβ1 (4 ng/ml, 24 h), as indicated. To detect CCN2 protein, equal amounts of cell layer extracts (20 μg) were subjected to Western blot analysis with anti-CCN2 antibody. Parallel blots were performed using an anti-GAPDH antibody. b C2 ceramide does not affect cell viability. Cells were treated with or without C2 ceramide as in a. An MTT assay was conducted to assess cell viability, as described in the “” section

C2 ceramide reduces TGFβ-induced CCN2 mRNA expression. Fibroblasts were incubated for 24 h in DMEM/0.5% FBS. Cells were then incubated with or without C2 ceramide (cer, 10 μΜ, 45 min) prior to incubation in the presence or absence of TGFβ1 (4 ng/ml, 6 h). RNA was harvested and subjected to real time RT-PCR and was normalized with ribosomal RNA. The experiment was performed in triplicate, on three independent occasions. A representative experiment is shown. The standard deviation within each data point was less than 10%. (*P < 0.001; significantly reduced mRNA due to inhibitor). Note that expression of both basal and TGFβ-induced CCN2 mRNAs were affected by C2 ceramide treatment

C2 ceramide reduces TGFβ-induced promoter activity (left panel) CCN2 promoter Fibroblasts were co-transfected with plasmids containing the wild-type CCN2 promoter driving the SEAP reporter gene and a CMV-β-gal construct. Cells were incubated for 18 h in DMEM/0.5% FBS, and treated with or without 10 μM C2 ceramide for 45 min followed by treatment with or without TGFβ1 for 24 h (4 ng/ml). SEAP expression values were measured in relative light units. The experiment was repeated performed twice. Values were expressed in relative light units and normalized with β-galactosidase. Values are mean of four replicates, ±standard deviation from three separate experiments. *P < 0.05; significantly different from control (DMSO alone). SBE-lux construct (right panel). Fibroblasts were co-transfected with plasmids containing multimers of a Smad binding element driving the luciferase reporter gene and a CMV-β-gal construct. Cells were incubated for 18 h in DMEM/0.5% FBS, and treated with or without C2 ceramide for 45 min (concentrations as indicated) followed by treatment with or without TGFβ1 for 24 h (4 ng/ml). Luciferase expression values were measured in relative light units. The experiment was repeated performed twice. Values were expressed in relative light units and normalized with β-galactosidase. Values are mean of four replicates, ±standard deviation from three separate experiments. *P < 0.05; significantly different from control (DMSO alone)