Fig 2: Ex Vivo Analyses Confirm the Anti-fibrotic Efficacy of the Hit Molecule in Human Fibrotic Samples(A) Fold change of gene expression in AA5-treated lung slice cultures (LSCs) relative to DMSO-treated controls at 48 h (n = 4). Data represent mean ± SEM. ***p < 0.001, **p < 0.01, and *p < 0.05 using two-way ANOVA and Sidak’s multiple comparison test.(B) Relative secreted levels of fibrosis-related proteins in supernatants of AA5-treated LSC cultures compared to DMSO-treated controls at 48 h (n = 9).(C) Relative secreted levels of HMGB1 in AA5-treated LSC cultures compared to DMSO-treated controls at 48 h (n = 9).(D) Representative image of DMSO- and AA5-treated LSC stained for HMGB1 counter stained for VIM and DAPI. Insets are higher magnification images; scalebar, 50 µm.(E) Representative FACS plots revealing expression of SSEA4+ population in LSCs treated with DMSO or AA5. Inset shows quantitative data of SSEA4+ cells. Data represent mean ± SEM. ***p < 0.001 using two-tailed paired t test.(F) ClueGO clustering analysis results of up- (red) and downregulated (blue) genes in a pairwise comparison of AA5- versus DMSO-treated LSCs (p % 0.05).(G and H) Fold change of expression of PTX3, NPTX1 and CD163L1 in AA5-treated LSCs relative to DMSO-treated controls at 48 h at mRNA (G; n = 6) and secreted protein (H; n = 6) levels.(I) Representative images from wells of the iFA model treated with the DMSO, 10 µM AA5, or 3.75 µg/ml IFN-? stained with Calcein AM and DAPI (left panel). Scale bars, top panel - 750 µm, bottom panel - 50 µm.(J) Relative fold change of secreted proteins in LSCs treated with 3.75 µg/ml rIFN-? for 48 h in comparison to DMSO-treated controls (n = 4).Data represent min-max and median protein abundance; ****p < 0.0001 ***p < 0.001 **p < 0.01 *p < 0.05 using two-way ANOVA and Sidak’s multiple comparison test.

Fig 3: Anti-inflammatory effect of FPF on bleomycin-treated skin. Immunohistochemical analysis of bleomycin-treated control (left panel) and FPF-treated (right panel) tissue samples were collected at Day 21 and stained with antibodies to CD3 to detect T-cells (A), MAC3 to detect macrophages (B), and CD163b to detect M2-polarized (C). Amelioration of dermal tissue inflammation in FPF-treated mice compared to control mice was observed, as indicated by the lower number of CD3-positive, MAC3-positive, and CD163b-positive cells. Representative images of five animals per group are shown. Scale bars = 200 µm.

Fig 4: Working model of peripheral‐type lung squamous cell carcinoma (pLSCC) development in the N‐nitroso‐tris‐chloroethylurea (NTCU)‐mouse model. Tumor associated macrophages (TAMs) that express CD163L1 and/or CD68 and/or CD204 might facilitate deltaNp63pos CD44vpos cancer cell expansion, invasion into surrounding alveoli, and immunosuppression through epidermal growth factor receptor (EGFR) and signal transducer and activator of transcription (STAT) signaling pathways in the peripheral portion of pLSCCs. Vertical arrows (red) indicate upregulation. LSH, Lymphoid‐specific helicase, ROS, reactive oxygen species.

Fig 5: AA5 Induces Acute-Phase Response Proteins Promoting Resolution of Fibrosis(A) Representative DMSO-treated, AA5-prevention (AA5p), and AA5-resolution (AA5r) iFA cultures immunostained for VIM and a-SMA (top panels) and collagen I and a-SMA (bottom panels). Scale bar, 50 µm.(B) Quantification of stiffness of the cells in the DMSO-treated, AA5p, and AA5r iFA model.(C) Secreted levels of HMGB1 in the iFA model during AA5p and AA5r treatments in comparison to DMSO-treated controls.(D and E) ClueGO clustering analysis results of up- (red) and downregulated (blue) genes in a pairwise comparison of AA5p (D) or AA5r (E) treatment with DMSO-treated iFA model. p values of = 0.05 are shown.(F) Cytokine profiles of acute-phase proteins in supernatants of the iFA model at day 13 relative to day 4 cytokines in AA5r or AA5p versus DMSO-treated cells(n = 4 in duplicate).(G) Cytokine profiles of proteins secondary to acute-phase response in supernatants from the iFA model at day 13 relative to day 4 cytokines in AA5r or AA5pversus DMSO-treated cells (n = 4 in duplicate).(H) Comparison of gene expression fold change levels of PTX3 and NPTX1 and scavenger receptor CD163L1 in the AA5r- and AA5p-treated iFA model when compared to the DMSO-treated iFA cultures (n = 5).(I) Time-dependent fold increase in secreted PTX3 and NPTX1 on AA5p and AA5r treatment measured in supernatants collected on day 13 (AA5p) and 48 h (AA5r) of treatment when compared to DMSO controls in the iFA model (n = 4).(J) Densitometric analysis depicting fold change of p-SMAD2/3 in the iFA model with DMSO, AA5-prevention, and AA5-resolution treatments analyzed by immunoblotting normalized to total protein.Data are presented as the mean ± SEM. ****p < 0.0001, ***p < 0.001, and **p < 0.01 using one- or two-way ANOVA and Dunnett or Sidaks’s multiple comparison test.