Fig. 9: Inhibition of the FACT complex counteracts fibrosis hallmarks in IPF.

a RNA-seq-based comparison of gene expression in IPF and after TGFB1 treatment. 2D Kernel Density plot representing the log2 fold change between gene expression in primary human lung fibroblasts (hLF) from IPF patients vs. control donors on the y-axis and log2 fold change between gene expression in Hmga2 + /+ MEF treated with TGFB1 vs. non-treated on the x-axis. Square, genes with log2 FC > 0.58 and P ≤ 0.05 in both, hLF IPF and TGFB1-treated MEF. P-values after Wald test. b Gene set enrichment analysis (GSEA) using the normalized enrichment scores (NES) of genes inside the square in a. EMT, epithelial-mesenchymal transition; resp, response. c GSEA line profile of the top two enriched pathways in b. d qRT-PCR-based expression analysis of selected HMGA2 target genes in hLF from control donors (Ctrl) or IPF patients that were non-treated (Ctrl) or treated FACT inhibitor (FACTin; CBLC000 trifluoroacetate) as indicated. e ChIP-based promoter analysis of selected HMGA2 target genes using pH2A.X-specific antibodies and chromatin from hLF treated as in d. f DIP-based promoter analysis of selected HMGA2 target genes using 5mC-specific antibodies and genomic DNA from hLF treated as in d. g qRT-PCR-based expression analysis of fibrotic markers in hLF treated as in d. FN1, fibronectin; COL1A1, collagen; ACTA2, smooth muscle actin alpha 2. h Functional assays for IPF hallmarks in Ctrl or IPF hLF treated as in d. Top, hydroxyproline assay for collagen content. Middle, proliferation assay by BrdU incorporation. Bottom, Transwell invasion assay. i Representative pictures from confocal microscopy after immunostaining using the antibody S9.6 or COL1A1-specific antibody in human precision-cut lung slices (hPCLS) from IPF patients (n = 3 biologically independent experiments). The hPCLS were treated as in d. DAPI, nucleus. Scale bars, 500 μm. In all bar plots, data are shown as means ± s.e.m. (n = 3 biologically independent experiments); asterisks, P-values after tow-tailed t-test, ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns, non-significant. See also Supplementary Fig. 10. Source data are provided as a Source Data files 01 and 04.