Fig 2: Effects of TA293 and mitoTA293 on oxidative stress, inflammation, and fibrosis in bleomycin (BLM)-induced pulmonary fibrosis model mice. Quantification of (a) oxidative stress and (b) inflammation in vivo. (c) Histopathology (hematoxylin and eosin (H&E) staining) and fibrosis (Masson’s trichrome staining) in mice 14 days after BLM administration. Pulmonary fibrogenesis was evaluated by semiquantitative analysis based on (d) Masson’s trichrome staining, (e) hydroxyproline content, and expression of (f) Acta2 and (g) Col1a1 mRNAs was determined using qRT-PCR. The data are presented as the mean ± SEM (n = 5 per group); ** p < 0.01; NS, not significant.

Fig 3: ATII cells undergoing RAS-induced EMT induce fibroblast activation via paracrine signalling. a Protein expression of a-SMA and phospho-Smad2 (p-Smad2) in MRC5 lung fibroblasts treated without or with 5 ng/ml TGFß in the presence of conditioned media (CM) from control or 4-OHT-treated ATIIER:KRASV12 cells for 48 h. ß-actin was used as a loading control. b Fold change in mRNA levels of COL1A1, COL3A1, FN1 and ACTA2 in MRC5 lung fibroblasts with indicated treatments. ß-actin-normalised mRNA levels in control cells were used to set the baseline value at unity. Data are mean ± s.d. n = 3 samples per group. *P < 0.05. ***P < 0.001. c Protein expression of a-SMA and phospho-Smad2 (p-Smad2), and fold change in the mRNA level of ACTA2 (a-SMA) in primary human lung fibroblasts from IPF (IPFFs) or from normal healthy lung (NHLFs) with indicated treatments. ß-tubulin was used as a loading control in Western blots. ß-actin-normalised mRNA levels in control cells were used to set the baseline value at unity (indicated above bars). Data are mean ± s.d. n = 3 samples per group. *P < 0.05. **P < 0.01. ***P < 0.001

Fig 4: Exosomes derived from HCT116-TP53(R273H) cells activate fibroblasts. (A) Western blot analysis of a-SMA and vimentin in mouse embryonic fibroblast (MEF) cells cocultured with conditioned medium derived from HCT116-TP53(R273H) and wild-type cells. GAPDH was used as a loading control; (B) Relative densitometry analysis of a-SMA expression normalized to that of GAPDH revealed a significant increase in protein levels after treatment with conditioned medium from HCT116-TP53(R273H) cells; (C) Western blot analysis of MEF cells cocultured with exosomes isolated from HCT116-TP53(R273H) cells; (D) Relative gene expression of ACTA2, VIM and TGFB2/3, encoding a-SMA, vimentin and TGF-ß2/3, respectively, by quantitative real-time polymerase chain reaction (qRT-PCR) in MEFs cocultured with exosomes derived from HCT116-TP53(R273H) cells for 1, 2, or 3 d. For B and D, triplicate biological repeats are displayed as \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\large \overline{{x}}{{±}}{{s}}_\overline{{x}}$\end{document}. WT, HCT116-TP53(WT); MT, HCT116-TP53(R273H); *, P<0.05; **, P<0.01; ***, P<0.001; (E) Fluorescence microscopy images of MEF cells treated with BODIPY-ceramide-labeled exosomes derived from the HCT116-TP53(R273H) cell line. Scale bars represent 20 µm; (F) Heat map of overlapping exosomal miRNAs; arrows indicate miR-769-3p and miR-21-3p. Red/green indicates an increase/decrease in gene expression. WT-Exo-1/2 and MT-Exo-1/2, duplicate biological repeats of exosomes extracted from HCT116-TP53(WT) and HCT116-TP53(R273H) cells, respectively.

Fig 5: Inhibition of HSC activation and the modulation of TGFß1 effects by XPO4. a) Ingenuity pathways analysis (IPA, Ingenuity systems, Inc., Redwood City, CA) revealed that hepatic fibrosis/hepatic stellate cell activation was one of the top significant overlapping pathway altered in XPO4 overexpressed cells compared to control. b) GSEA analysis similarly found significant enrichment in gene sets associated with extracellular matrix and collagen-related pathways in control cells compared to XPO4 overexpressed cells c) Heat map of fold change of genes involved in fibrosis in murine JS-1 HSCs in Xpo4 overexpressed cells compared to control for 24 hours followed by TGFß1 (2.5 ng/ml) treatment or vehicle control for 24 h using log2-transformed mRNA-Seq expression data, n = 4 per group. d) Acta2, Col1a1, Ctgf, Tgfb1, Mmp9 and Timp1 qRT-PCR analysis in murine HSCs transfected with control or Xpo4 vector for 24 hours followed by TGFß1 (2.5 ng/ml) or vehicle control treatment for 24 h. e) RT-PCR analysis for the same panel of genes in murine HSCs transfected with control or Xpo4 siRNA for 24 hours followed by TGFß1 (2.5 ng/ml) or vehicle control treatment for 24 h. f) COL1A1 level as measured by Sircol™ Collagen Assay, g) Acta2 expression as assessed by immunofluorescence and h) measured fluorescence intensity and i) ROS generation as measured by fluorescence intensity was significantly reduced in Xpo4-vector-transfected JS-1 cells treated with or without TGFß1 (2.5 ng/mL for 24 h) compared to control. Results are expressed as mean ± SEM of at least three independent experiments performed in triplicate; statistical significance of the differences between groups was calculated using the unpaired two-sample Student's t-test or ANOVA test. *P < 0.05, ** P<0.01, *** P<0.001.