Fig 2: Normal lung fibroblasts were grown on tissue culture plastic in DMEM supplemented with 5% FBS to 60% confluence, serum deprived for 24 h in DMEM without serum and then treated with/without TGF-ß1 at the indicated doses and durations. a and b Fas mRNA determined by quantitative real-time RT-PCR (qPCR); pooled data with an “n” of at least 7 independent replicates for each dose and time point. c and d Representative Western blots for Fas protein in whole cell lysates, with densitometric analysis. Densitometry represents data with at least 7 independent experimental replicates for each dose at the 24-h time point and 4 independent replicates at the 4- and 8-h time points. * p < 0.05 and ** p < 0.01 compared to untreated controls (One-way ANOVA with Tukey multiple comparison test). e and f Fas protein in whole cell lysates measured by ELISA. Data represent pooled results from at least 5 independent experimental replicates at the 24-h time points and 2–3 experimental replicates at the 4- and 8-h time points. ** p < 0.01 compared to control (One-way ANOVA with Tukey multiple comparison test). g Representative Western blot and (h) densitometry for Fas. n = at least 5 independent experimental replicates for each condition. *** p < 0.001 compared to untreated controls (One-way ANOVA with Tukey multiple comparison test)

Fig 3: Normal lung fibroblasts were pre-treated with/without the FAK inhibitor PF573228 (10 µM) or the PI3K inhibitor LY294002 (10 µM) for one hour prior to treatment with TGF-ß1 (10 ng/ml). Fas expression was then assessed by (a) qPCR; * p < 0.05 compared to untreated controls, n = 2 independent experimental replicates, (b) Western blot of whole cell lysates; representative blot with densitometry from three independent experimental replicates. * p < 0.05 and ** p < 0.01 compared to untreated controls

Fig 4: a Normal lung fibroblasts in serum-free media were treated with the combination of TNF-a (10 ng/ml) and IFN-? (50 U/ml) for the indicated times up to 48 h. For the groups receiving inflammatory cytokines for 6–24 h, at the end of the exposure cells were washed with PBS and replaced in serum free media. After 48 h, the cells were washed again with PBS and placed in serum free media with/without TGF-ß1 (10 ng/ml) for another 24 h. Whole cell lysates were then assessed for Fas expression. Additional experiments were done to confirm the effects at 24 and 48 h, and the densitometry represents three independent experiments at those time points. b Normal lung fibroblasts were treated with/without TNF-a (10 ng/ml) and IFN-? (50 U/ml) for 24 h, washed with PBS and then exposed to Fas-activating antibody (Fas-Ab; 250 ng/ml) with/without TGF-ß1 (10 ng/ml) for 16 h. Whole cell lysates were assessed for PARP. Lane 7 is from control fibroblasts treated with the Fas-activating antibody in combination with cycloheximide (CHX; 500 ng/ml) as a positive control for apoptosis

Fig 5: a Normal lung fibroblasts were cultured on polyacrylamide substrates with Young’s moduli of 400 Pa or 6400 Pa for 24 h in serum-free media prior to treatment with/without the Fas-activating antibody (Fas-Ab, 250 ng/ml) for 16 h, and apoptosis was assessed using ELISA detection of histone-associated DNA-fragments. The figure represents pooled data from five independent experiments with 3 technical replicates included in each experiment, with the data for each independent experiment normalized to 400 Pa controls to allow comparisons. *** p < 0.001 compared to untreated cells on 400 Pa. Comparisons were made using one-way ANOVA with Tukey’s multiple comparison test. b Fas mRNA in normal fibroblasts cultured for 24 h on 400 or 6400 Pa substrates was assessed by quantitative real-time RT-PCR. n = 3 independent experiments with two technical replicates per experiment, with data normalized to the average expression in cells on the 6400 Pa substrates. * p < 0.05 using unpaired T-test. c Fas protein in cell lysates was measured by ELISA. Data pooled from 3 independent experiments. Fas concentration was determined as pg Fas/µg protein collected and was then normalized to the average concentration of 6400 Pa substrates to allow direct comparisons between experimental replicates. * p < 0.05 using unpaired T-test