Fig. 8.

ATP-competitive mTOR inhibition attenuates TGF-β₁-induced matrisome protein expression in vitro and collagen synthesis in IPF lung slices. IPF human lung fibroblasts (IPF-HLFs) were pre-incubated with vehicle (0.1% DMSO), rapamycin (100 nM) or the mTOR inhibitor CZ415 (5 µM) prior to stimulation with TGF-β₁ (1 ng/ml) for 24 h. Proteomic analysis representing fold change (FC) of core matrisome proteins and proteins involved in collagen synthesis and degradation in lysates of TGF-β₁ + vehicle/drug-treated fibroblasts versus negative TGF-β₁ control is displayed in a heatmap. Only proteins with up- or down-regulation (pAdj < 0.05 and |FC| > 1.2 for both replicates) in any treatment versus negative TGF-β₁ control are represented (50 proteins) (a). Quantitative profiles across treatments are displayed as line charts (b). Human IPF lung tissue slices generated from IPF lung transplant tissue were treated with vehicle (0.1% DMSO) or increasing concentrations of CZ415 for 120 h. Levels of P1NP in supernatants from 4 slices per condition were assessed by ELISA (c). Data are representative of 5 independent donors. Differences between conditions were evaluated with one-way ANOVA with Dunnett’s multiple comparison testing, **p < 0.005. Human IPF lung tissue slices (n = 4) from 2 donors were treated with vehicle (0.1% DMSO) or CZ415 (1 μM) for 5 days and P1NP levels in supernatants and phosphoproteins in homogenized slices were measured. Pooled donor data are presented in d. Differences between conditions were evaluated with Student’s t-test, **p < 0.005, ****p < 0.0001. In c, d the boxplot center line, bounds of box, and whiskers represent median, inter-quartile range, and minimum to maximum values