Fig 2: PIM1 and NFATc1 signaling pathways are associated with impaired fibrosis resolution in aged mice after bleomycin injury.(A) Venn diagrams of differentially regulated genes from uninjured aged and injured aged fibroblasts relative to uninjured young fibroblasts. Intersection genes with the highest average expression are displayed. (B) Ingenuity pathway analysis of the intersection genes identifies differentially regulated canonical pathways. (C) De novo transcription factor binding motif analysis of differentially expressed genes in young fibroblasts after injury showing the top 3 binding motif and their associated transcription factors. (D) De novo transcription factor binding motif analysis of differentially expressed genes in aged lung fibroblasts after injury showing the top 3 binding motif and their associated transcription factor. Red box indicates highest ranked transcription factor binding motif and associated transcription factor in aged lung fibroblasts. (E) Heatmap of genes with NFATc1 binding motifs displayed as Z scores of RPKM (young sham, n = 2; young bleo 30 days, n = 5; aged sham, n = 4; aged bleo 30 days, n = 5). (F) Expression of PIM1 gene in young and aged lung fibroblasts (young sham, n = 2; aged sham, n = 4). (G) STRING functional protein association networks showing an interconnection between PIM1, STAT3, and NFATc1. Each node represents a protein, and each line represents a curated interaction.

Fig 3: PIM1 inhibition inhibits profibrotic gene expression and collagen secretion in organotypic IPF lung cultures ex vivo.(A) Schematic of organotypic culture of IPF lungs. (B) H&E and trichrome staining of IPF lung tissue explants after 5 days in culture showing intact tissue architecture of nonfibrotic area (top panels) and distorted architecture of fibrotic area (bottom panels). Scale bar: 50 µm. FF, fibrotic foci; V, vein; Av, alveoli. (C) qPCR analysis of ECM gene expression in IPF lung explants treated with 10 µM of AZD1208 or DMSO control in combination with or without 10 ng/mL of TGF-ß for 5 days. n = 3 IPF lung explants. Data are shown as mean ± SEM. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (D) qPCR analysis of pathogenic lung fibroblasts gene markers in IPF lung explants treated with 10 µM of AZD1208 or DMSO in the presence or absence of 10 ng/mL of TGF-ß for 5 days. n = 3 IPF lung explants. Data are shown as mean ± SEM. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (E) qPCR analysis of prosurvival genes in IPF lung explants treated with 10 µM of AZD1208 or DMSO in the presence or absence of 10 ng/mL of TGF-ß for 5 days. n = 3 IPF lung explants. Data are shown as mean ± SEM. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (F) Soluble collagen-I secreted from IPF lung explants into the media was evaluated by Western blot analysis. Each lane contained equal volume of conditioned medium of different lung sections obtained from single IPF lung explants. Ponceau staining was used as loading control for secreted collagen-I.

Fig 4: Inhibition of PIM1 signaling pathway reduces profibrotic gene expression in IPF-derived lung fibroblasts.(A) qPCR analysis of profibrotic gene expression in IPF-derived lung fibroblasts transfected with scrambled siRNA or siRNA for NFATc1 for 48 hours, followed by treatment with 2 ng/mL of TGF-ß for an additional 24 hours. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (B) IPF-derived lung fibroblasts were transfected with scrambled or NFATc1 siRNAs for 48 hours and then treated with 2 ng/mL of TGF-ß for an additional 24 hours, followed by Western blotting analysis. Shown is a representative blot of 3 independent experiments. (C) qPCR analysis of profibrotic gene expression in IPF-derived lung fibroblasts treated with VIVIT (5 µM) and 2 ng/mL of TGF-ß for 24 hours. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (D) IPF-derived lung fibroblasts were treated with VIVIT (5 µM) and 2 ng/mL of TGF-ß for 24 hours and analyzed via Western blot. Shown is a representative blot of n = 2 independent experiments. (E) qPCR analysis of profibrotic genes in IPF-derived lung fibroblasts cotreated with 10 µM of AZD1208 and 2 ng/mL of TGF-ß for 24 hours. Data are shown as mean ± SEM of n = 3 independent experiments. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (F) IPF-derived lung fibroblasts were cotreated with 10 µM of AZD1208 and 2 ng/mL of TGF-ß for 24 hours and analyzed by Western blotting. Shown is a representative blot of 3 independent experiments. (G) IHC of IPF-derived fibroblasts cotreated with 10 µM of AZD1208 and 2 ng/mL of TGF-ß for 24 hours. Scale bar: 50 µm. Representative images of 3 independent experiments are shown.

Fig 5: Inhibition of PIM1 signaling pathway reduces prosurvival gene expression and sensitizes IPF-derived lung fibroblasts to apoptotic cues.(A) qPCR analysis of aging-associated prosurvival genes and those with NFATc1 binding sites in IPF-derived fibroblasts transfected with scrambled siRNA or siRNA for NFATc1 for 48 hours. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using 2-tailed, paired Student’s t test. (B) Western blot analysis of FOXM1 and BIRC5 in IPF-derived fibroblasts transfected with scrambled siRNA or siNFATc1 for 48 hours and then stimulated with 50 ng/mL of PDGF-BB or 10% FBS for an additional 24 hours. Shown is a representative blot of 2 independent experiments. (C) qPCR analysis of prosurvival genes in IPF-derived fibroblasts transfected with scrambled siRNA or siRNA for PIM1 for 48 hours. Data are shown as mean ± SEM of n = 4 independent experiments. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (D) qPCR analysis of prosurvival gene expression in IPF-derived lung fibroblasts cotreated with 10 µM of AZD1208 and 50 ng/mL of PDGF-BB for 24 hours. Data are shown as mean ± SEM of n = 3 independent experiments. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (E) IPF-derived lung fibroblasts were cotreated with 10 µM of AZD1208 and 50 ng/mL of PDGF-BB for 24 hours and analyzed by Western blotting using antibodies against phospho-BAD, total-BAD, BIRC5, and GAPDH. (F) IPF-derived lung fibroblasts were pretreated for 24 hours with 10 µM of AZD1208 and with 300 nM of staurosporine for an additional 2 hours, followed by Western blotting analysis using antibodies against procaspase-3, cleaved caspase-3, and GAPDH. (G) IPF-derived lung fibroblasts were preincubated for 24 hours with 10 µM of AZD1208, followed by treatment with 0.5 µg/mL of FAS-activating antibody for an additional 24 hours. Procaspase-3 and cleaved caspase-3 were detected by Western blotting. Shown is a representative blot of 3 independent experiments.