Fig 2: Extracellular matrix forming proteins are highly expressed in IPF.A–C, violin plots showing mRNA of a-SMA, Col1a1, and Fibronectin in Gene Expression Affymetrix data (GSE 150910) of lungs of control donor and IPF patients. Data presented as means ± s.e.m., n = 102 to 103, *p < 0.05 (Student’s t-test). D, immunofluorescence staining of NNMT in lung sections from control and IPF patients. NNMT -red; nuclei -blue. Scale bar 50 µm. E, fluorescence DSP images of control donor and IPF lung tissues showing a-SMA expression in red outlined Regions of Interest (ROI). Pan-keratin (green), CD31 (red), a-SMA (yellow), nucleus (blue). Scale bar 200 µm. F, heatmap showing comparative quantitation of a-SMA in ROIs of control and IPF lung tissues. G–I, mRNA of a-SMA, Col1a1, and Fibronectin (FN1) in Digital Spatial Profiling (DSP) representing six Regions of Interest (ROIs) from lung sections of control and individual with IPF; n = 6 representing one individual, each with analysis of six random Regions of Interest (ROIs). The data are presented as means ± s.e.m., *p < 0.05 (Student’s t-test).

Fig 3: NNMT regulates extracellular matrix production, cellular proliferation, and metabolic pathways in lung fibroblasts.A–H, Western blot and quantitative analysis of protein expression of NNMT, Col1a1, FN, SIRT3, ACLY, PCNA, and Cyclin D1 in IMR-90 fibroblasts transfected with non-targeting (NT) or NNMT siRNA (100 nM) for 72 h. Data presented as means ± s.e.m., n = 3, *p < 0.05 (Student’s t test). I–L, fold change in mRNAs of NNMT, ACTA2, Col1a1, FN1, and ACLY in RNA-seq analysis of human fibroblasts transfected with non-targeting or NNMT siRNA (100 nM) followed by treatment with TGF-ß1 (0 or 2.5 ng/ml) for 48 h. Data presented as means ± s.e.m., n = 3, One-way ANOVA.

Fig 4: NNMT regulates lipofibroblast-myofibroblast phenotypic transition.A–D, violin plots showing mRNA of PPAR?, TCF21, Acetyl-CoA-Carboxylase (ACAC), and Perilin2 in Gene Expression Affymetrix data (GSE150910) of lungs of control donor and IPF patients. Data presented as means ± s.e.m., n = 102 to 103, *p < 0.005 (Student’s t test). E-G, mRNA of PPAR?, TCF21, and Perilin2 in RNA-seq analysis of human fibroblasts transfected with non-targeting or NNMT siRNA (100 nM) followed by treatment with TGF-ß1 (0 or 2.5 ng/ml) for 48 h. Data presented as means ± s.e.m., n = 3, One-way ANOVA. H–K, Western blot and quantitative analysis of NNMT, PPAR?, and TCF21 in whole cell lysates of IMR-90 fibroblasts transfected with NT or NNMT siRNA (100 nM) for 72 h followed by treatment with vehicle or TGF-ß1 (2.5 ng/ml) for 36 h. Data presented as means ± s.e.m., n = 3, One-way ANOVA. L, flow cytometric analysis of control and IPF fibroblasts stained with APC-labelled NNMT and FITC-labelled PPAR?. Cells were first gated for FSC and SSC followed by NNMT-APC and PPAR? -FITC. M, plots show the percentage of population positive for NNMT-APC (grouped quadrant B2) and PPAR?-FITC (grouped quadrant B4) from (L). Results represent data from n = 3 representing fibroblasts of three donor and IPF subjects each. Data presented as means ± s.e.m., n = 3, ***P = <0.0002 (Student’s t test).

Fig 5: NNMT mediates TGF-ß1 induced anti-apoptotic phenotype in human lung fibroblasts.A, Western blot showing expression of NNMT in IMR-90 fibroblasts transfected with NT or NNMT siRNA (100 nM) for 72 h followed by treatment with vehicle or TGF-ß1 (2.5 ng/ml) for 36 h. B, flow cytometric analysis of fibroblasts stained with Annexin V and Propidium iodide after transfection with NT or NNMT siRNA (100 nM) for 72 h followed by treatment with vehicle or TGF-ß1 (2.5 ng/ml) for 36 h.