Expression of cyclin D1 mRNA and protein in human lung fibroblasts; response to fibrogenic growth factors. Figure 2a: Cyclin D1 mRNA levels were determined by quantitative real-time PCR on three separate human lung fibroblast cell lines from IPF patients (HIPF, LL29, and LL97a) and normal control equivalents CCD8LU exposed to fibrogenic mediators TGF-β1 (5 ng/ml and 10 ng/ml) and CTGF (10 ng/ml and 100 ng/ml) for 8 hrs. Data shown demonstrates analysis from LL97a and CCD8LU fibroblasts, no significant difference was observed in baseline cyclin D1 expression between the cell lines. Data are representative of 3 independent experiments, within each of which PCRs were performed in triplicate. Data represents mean cyclin D1 expression ± S.E.M; * = p < 0.05 compared to serum free, † = p < 0.05 compared to normal lung fibroblasts. Figure 2b: Quantification of cyclin D1 protein expression was performed by western blotting in all three IPF fibroblast lines and normal equivalents. Quiescent serum deprived lung fibroblasts were stimulated with fibrogenic growth factors for 24 hours. Cyclin D1 protein levels found in 25 μg of total protein from normal and IPF derived lung fibroblasts was determined by western blotting. Data shown demonstrates analysis from LL97a and CCD8LU fibroblasts. Data are representative of 3 independent westerns and represented as mean density ± SEM. * = p < 0.05 compared to normal lung fibroblasts. Figure 2c: Representative western blots for cyclin D1 and GAPDH protein expression in a representative IPF lung fibroblast cell line (LL97a). (i) cyclin D1 blot-lane 1 = marker lane 2 = control (serum deprived); lane 3 = 10% FCS; lane 4 = TGF-β1 1 ng/ml; lane 5 = TGF-β1 5 ng/ml; lane 6 = CTGF 10 ng/ml; lane 6 = CTGF 100 ng/ml. (ii) GAPDH blot-lane 1 = control (serum deprived); lane 2 = 10% FCS; lane 3 = TGF-β1 1 ng/ml; lane 4 = TGF-β1 5 ng/ml; lane 5 = marker; lane 6 = CTGF 10 ng/ml; lane 6 = CTGF 100 ng/ml. Ponceau S staining of blots after transfer revealed equivalent loading of total protein.