Fig. 9

mTORC1/4E-BP1 axis mediates collagen I deposition in other mesenchymal cells. Lung adenocarcinoma-associated fibroblasts (CAFs), primary human dermal fibroblasts (pHDFs), and primary hepatic stellate cells (HSCs) 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 (a, e, i). CRISPR-Cas9-edited CAFs, pHDFs, and HSCs were stimulated with TGF-β₁ (1 ng/ml) for 72 h, with collagen I deposition assessed by macromolecular crowding assay (b, f, j). Data are expressed as collagen intensity (n = 4 fields of view imaged per well) normalized to cell count. Data are presented as mean ± SEM (CAFs n = 5, pHDFs n = 6, HSCs n = 8). CAFs, pHDFs, and HSCs were transfected with control siRNA or siRNA targeting 4E-BP and 4E-BP1 protein expression was measured (c, g, k). Following transfection, cells were preincubated with 1 μM (CAFs) or 300 nM (pHDFs, HSCs) AZD8055 or vehicle prior to stimulation with TGF-β₁ for 72 h. Collagen I deposition was analyzed by macromolecular crowding assay (d, h, l). Data are expressed as collagen intensity (n = 4 fields of view imaged per well) normalized to cell count. Data are presented as mean ± SEM (CAFs n = 3, pHDFs n = 4, HSCs n = 5). Differences between groups were evaluated with two-way ANOVA with Tukey multiple comparison testing, *p < 0.05, ***p < 0.001, ****p < 0.0001