Fig. 7.

mTORC1 plays a critical role in mediating the pro-fibrotic effects of TGF-β₁ in IPF-derived lung fibroblasts. IPF human lung fibroblasts (IPF-HLFs) were pre-incubated with vehicle (0.1% DMSO) or increasing concentrations of AZD8055 (a), CZ415 (b), or rapamycin (c) and stimulated with TGF-β₁ (1 ng/ml) for 72 h with collagen I deposition assessed by macromolecular crowding assay. Data are expressed as collagen I signal calculated as a percentage of the TGF-β₁-treated control (n = 4 fields of view imaged per well) and cell counts obtained by staining nuclei with DAPI. Each data point shown is mean ± SEM (n = 4) and is representative of 5 independent experiments. IC₅₀ values were calculated using 4-parameter non-linear regression: AZD8055, IC₅₀ = 604 nM, 95% CI 415 nM to 1 µM; CZ415, IC₅₀ = 245.3 nM, 95% CI 136.7–541 nM. Additionally, IPF-HLFs were modified by CRISPR-Cas9 gene editing using gRNAs targeting exon 26 of RPTOR or exon 29 of RICTOR. Analysis of the resultant levels of Raptor and Rictor protein are shown (d). CRISPR-Cas9-edited IPF-HLFs were also stimulated with TGF-β₁ (1 ng/ml) for 72 h, with collagen I deposition assessed by macromolecular crowding assay (e). Scale bars = 360 µm