Fig. 1

Anti-fibrotic effects of AZT in control and IPF fibroblasts. Isolated control human lung fibroblasts (n = 5) and IPF fibroblasts (n = 5) were treated with resting media (RM) for 24 h, 50 μM AZT, 5 ng/ml TGF-β or co-incubated with TGF-β + AZT (T + A). Pro-fibrotic gene expression was determined by RT-qPCR (a: Col1A1, b: FN). Data represents fold increase relative to the negative control (RM). One-way ANOVA followed by Tukey’s post test was used for statistical analysis. Protein expression was determined by ELISA (c: pro-Col1A1) and one-way ANOVA followed by Bonferroni’s post test was used for statistical analysis. For western blot analysis (d: FN; f: αSMA) one-way ANOVA followed by Tukey’s post was used to analyze data. a Col1A1 gene expression was significantly reduced in control and IPF fibroblasts after AZT and TGF-β treatment compared to TGF-β alone (**p-value ≤0.01, ***p-value ≤0.001). b FN gene expression was not significantly altered after AZT neither in control nor in IPF fibroblasts compared to TGF-β alone. c Collagen (pro-Col1A1) production was measured by ELISA in cell culture supernatant from control (n = 4) and IPF fibroblasts (n = 5). Additional AZT treatment induced a significant reduction in collagen secretion compared to TGF-β treatment alone in IPF fibroblasts, but not in control fibroblasts (*p-value ≤0.05). Combined data is presented and data was normalized to β2M, and means (+/− SEM) are presented as fold increase relative to the negative control RM. Combined data were analyzed. d FN protein expression was assessed by western blot analysis in whole cell lysates of control and IPF fibroblasts. FN protein expression was significantly reduced after AZT and TGF-β treatment in IPF fibroblasts when directly compared to controls (*p-value ≤0.05). Combined quantified data of control and IPF patients (n = 4) relative to RM are shown. e Immunofluorescent staining of IPF fibroblasts visually revealed that αSMA fiber expression (red) was reduced after AZT treatment compared to TGF-β alone and induced vacuolar formations. Magnification of 20x. Scale bar = 25 μM. f αSMA protein expression was significantly reduced only in IPF fibroblasts after AZT and TGF-β treatment compared to TGF-β alone (***p-value ≤0.001). Combined quantified data of control and IPF patients (n = 4) relative to RM are shown. At least three independent experiments were performed