Figure 5
HBE cells conditioned medium induces fibroblast proliferation and profibrotic gene expression. A) Experimental protocol for conditioned media experimentation. HBE cells were treated with different doses of MWCNT (12, 24 μg/mL equivalents 3.6 μg/cm2) for 24 hours, cell culture supernatants were removed, centrifuged and diluted 1:4 with DMEM/F-12 medium (without serum). Fibroblasts were grown till near confluence and serum starved for 24 hours then exposed to conditioned medium from epithelial cells for 48 hours. mRNA was extracted from these cells to perform pro-fibrotic gene expression analysis using real time quantitative RT-PCR. B) Fibroblast proliferation assay after CM exposure for 48 hours. TGF-β (10 ng/mL) was used as a positive control. C) Dose response analysis of TIMP-1, TN-C, PC-1, TGF-β and OPN expression in MRC-5 cells after CM exposures. Data were analyzed by analysis of variance (ANOVA) followed by Tukey’s post hoc test. Graphs show average ± SEM of three independent experiments with triplicate of each condition, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (between media-treated control and treatment).