Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: IPF Pathogenesis is Dependent upon TGFβ Induction of IGF-1

doi: 10.1096/fj.201901719RR

Figure Lengend Snippet: Summary of cell culture treatments.

Article Snippet: Antigen specific primary antibodies to IGF-1 (4 μg/mL, AB9572; ABCAM, Cambridge, MA, USA) or p-IGF-1R (1:50 dilution, NB100–92555; Novus Biologicals, Centennial, Colorado, USA) were used by overnight 4°C incubation in a hydration container, counterstained with Mayer’s hematoxylin (Gills Formula #2, CS401–1D; ThermoFisher Scientific, Waltham, MA), and dehydrated with ethanol and Clear-Rite 3 (#6901; ThermoFisher Scientific, Waltham, MA) before mounting with non-aqueous VectaMount (H-5000; Vector Laboratories, Burlingame, California, USA).

Techniques: Cell Culture, Concentration Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: IPF Pathogenesis is Dependent upon TGFβ Induction of IGF-1

doi: 10.1096/fj.201901719RR

Figure Lengend Snippet: Summary of antibodies used for Western blot and immunoprecipitation.

Article Snippet: Antigen specific primary antibodies to IGF-1 (4 μg/mL, AB9572; ABCAM, Cambridge, MA, USA) or p-IGF-1R (1:50 dilution, NB100–92555; Novus Biologicals, Centennial, Colorado, USA) were used by overnight 4°C incubation in a hydration container, counterstained with Mayer’s hematoxylin (Gills Formula #2, CS401–1D; ThermoFisher Scientific, Waltham, MA), and dehydrated with ethanol and Clear-Rite 3 (#6901; ThermoFisher Scientific, Waltham, MA) before mounting with non-aqueous VectaMount (H-5000; Vector Laboratories, Burlingame, California, USA).

Techniques: Western Blot, Immunoprecipitation

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: IPF Pathogenesis is Dependent upon TGFβ Induction of IGF-1

doi: 10.1096/fj.201901719RR

Figure Lengend Snippet: (A) RT-qPCR of Igf-1 gene expression in AKR-2B murine fibroblasts treated with TGFβ compared to vehicle over 24 hours (n=3). (B) RT-qPCR of Igf-1 gene expression in whole lung tissue from intratracheal Bleomycin (BLM, n=5) treated mice compared to Saline (n=5). (C) Western blot and (D) ELISA analysis of IGF-1 ligand expression/secretion following TGFβ or vehicle treatment in AKR-2B fibroblasts over 24 hours (n=3 for both). (E) IGF-1 receptor (IGF-1R) was immunoprecipitated (IP) from AKR-2B murine fibroblasts at the indicated times following treatment with TGFβ (+) or vehicle (−) and Western blotted (WB) for p-IGF-1R (n=3). Data are presented as means −/+ Standard Error of the Mean (SEM) for the number of biological replicates indicated (n). Statistical significance was determined after computing single factor ANOVA and/or unpaired two-tailed Student’s t-test (p<0.05 (*), p<0.01 (**), p<0.005 (***), p<0.001 (****). Results demonstrate that TGFβ stimulates Igf-1 gene expression, ligand production, and IGF-1R activation in murine fibroblasts.

Article Snippet: Antigen specific primary antibodies to IGF-1 (4 μg/mL, AB9572; ABCAM, Cambridge, MA, USA) or p-IGF-1R (1:50 dilution, NB100–92555; Novus Biologicals, Centennial, Colorado, USA) were used by overnight 4°C incubation in a hydration container, counterstained with Mayer’s hematoxylin (Gills Formula #2, CS401–1D; ThermoFisher Scientific, Waltham, MA), and dehydrated with ethanol and Clear-Rite 3 (#6901; ThermoFisher Scientific, Waltham, MA) before mounting with non-aqueous VectaMount (H-5000; Vector Laboratories, Burlingame, California, USA).

Techniques: Quantitative RT-PCR, Gene Expression, Saline, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Immunoprecipitation, Two Tailed Test, Activation Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: IPF Pathogenesis is Dependent upon TGFβ Induction of IGF-1

doi: 10.1096/fj.201901719RR

Figure Lengend Snippet: (A, B, D) Representative images (20x magnification) from histological sections of murine lung tissue with the following treatments: intratracheal Bleomycin (BLM, left, n=4), Saline (center, n=3), and Bleomycin dual-treated with Imatinib and Lapatinib (BIL, right, n=3; previously shown to be effective in treating BLM-induced fibrosis [23]) were stained with Masson’s Trichrome (A, top) to visualize lung tissue architecture (purple, nuclear; pink, cytoplasmic) and collagen composition (blue, collagen fibers), anti-IGF-1 antibodies (B, middle), anti-phosphorylated-IGF-1R antibodies (D, bottom) co-stained with Hematoxylin, or negative controls (NEG) without primary antibody co-stained with Hematoxylin. Color deconvoluted images (B, D) show the intensity of IGF-1 ligand or phosphorylated-IGF-1R present in BLM mice compared to Saline and BIL per the deconvolution color key and quantified using a pixel count algorithm (C, E). Data are presented as means −/+ Standard Error of the Mean (SEM) for the number of biological replicates indicated (n). Statistical significance was determined after computing single factor ANOVA and/or unpaired two-tailed Student’s t-test (p<0.05 (*), p<0.01 (**), p<0.005 (***), p<0.001 (****). Results demonstrate that IGF-1 ligand and IGF-1R activation are increased in fibrotic murine lung tissue and diminished following anti-fibrotic treatment.

Article Snippet: Antigen specific primary antibodies to IGF-1 (4 μg/mL, AB9572; ABCAM, Cambridge, MA, USA) or p-IGF-1R (1:50 dilution, NB100–92555; Novus Biologicals, Centennial, Colorado, USA) were used by overnight 4°C incubation in a hydration container, counterstained with Mayer’s hematoxylin (Gills Formula #2, CS401–1D; ThermoFisher Scientific, Waltham, MA), and dehydrated with ethanol and Clear-Rite 3 (#6901; ThermoFisher Scientific, Waltham, MA) before mounting with non-aqueous VectaMount (H-5000; Vector Laboratories, Burlingame, California, USA).

Techniques: Saline, Staining, Two Tailed Test, Activation Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: IPF Pathogenesis is Dependent upon TGFβ Induction of IGF-1

doi: 10.1096/fj.201901719RR

Figure Lengend Snippet: (A, C) RT-qPCR or (B, D) Western blot analysis of Extracellular Matrix (ECM) genes/proteins in AKR-2B murine fibroblasts (A, B) or MRC5 human lung fibroblasts (C, D) pre-treated for 2 hours with increasing concentrations of OSI-906 or vehicle (0.1% DMSO) followed by stimulation with TGFβ for 24 hours (n=3). ECM genes/proteins included in the transcriptional/translational profiles regulated by OSI-906 include: Alpha Smooth Muscle Actin, ACTA2/Acta2 (αSMA); Connective Tissue Growth Factor, Ctgf; Collagen Type 5 Alpha 3 Chain, COL5A3/Col5α3; Plasminogen Activator Inhibitor-1, PAI-1/Pai-1; Collagen Type I Alpha 1 Chain, COL1A1/Col1α1; Collagen Type 7 Alpha 1 Chain, COL7A1; Extra Domain-A Fibronectin, EDA-FN/Eda-fn. (A, C) Relative fold gene induction by TGFβ compared to vehicle is indicated at the top of each control bar. Data are presented as means −/+ Standard Error of the Mean (SEM) for the number of biological replicates indicated (n). Statistical significance was determined after computing single factor ANOVA and/or unpaired two-tailed Student’s t-test (p<0.05 (*), p<0.01 (**), p<0.005 (***), p<0.001 (****). Results demonstrate that OSI-906 inhibition of IGF-1R kinase activity is effective in reducing the expression of profibrotic ECM components induced by TGFβ.

Article Snippet: Antigen specific primary antibodies to IGF-1 (4 μg/mL, AB9572; ABCAM, Cambridge, MA, USA) or p-IGF-1R (1:50 dilution, NB100–92555; Novus Biologicals, Centennial, Colorado, USA) were used by overnight 4°C incubation in a hydration container, counterstained with Mayer’s hematoxylin (Gills Formula #2, CS401–1D; ThermoFisher Scientific, Waltham, MA), and dehydrated with ethanol and Clear-Rite 3 (#6901; ThermoFisher Scientific, Waltham, MA) before mounting with non-aqueous VectaMount (H-5000; Vector Laboratories, Burlingame, California, USA).

Techniques: Quantitative RT-PCR, Western Blot, Control, Two Tailed Test, Inhibition, Activity Assay, Expressing

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: IPF Pathogenesis is Dependent upon TGFβ Induction of IGF-1

doi: 10.1096/fj.201901719RR

Figure Lengend Snippet: (A, B, D) Representative images (30x magnification) from histological sections of control (right, n=7, areas of normal human lung enriched with fibroblasts) and fibrotic (left, n=12, IPF fibroblastic foci) patient tissue were stained with Masson’s Trichrome (A, top) to visualize lung tissue architecture (purple, nuclear; pink, cytoplasmic) and collagen composition (blue, collagen fibers), anti-IGF-1 antibodies (B, middle), anti- phosphorylated-IGF-1R antibodies (D, bottom) co-stained with Hematoxylin, or negative controls without primary antibody co-stained with Hematoxylin (NEG). Color deconvoluted images (B, D) show the area and intensity of IGF-1 ligand (B) or phosphorylated-IGF-1R (D) present in regions of human lung enriched with proliferating myofibroblasts in fibroblastic foci compared to fibroblasts in supportive pulmonary structures per the deconvolution color key and quantified using a pixel count algorithm (C, E). Data are presented as means −/+ Standard Error of the Mean (SEM) for the number of biological replicates indicated (n). Statistical significance was determined after computing single factor ANOVA and/or unpaired two-tailed Student’s t-test (p<0.05 (*), p<0.01 (**), p<0.005 (***), p<0.001 (****). Results demonstrate that IGF-1 ligand and IGF-1R activation are increased in IPF fibroblastic foci.

Article Snippet: Antigen specific primary antibodies to IGF-1 (4 μg/mL, AB9572; ABCAM, Cambridge, MA, USA) or p-IGF-1R (1:50 dilution, NB100–92555; Novus Biologicals, Centennial, Colorado, USA) were used by overnight 4°C incubation in a hydration container, counterstained with Mayer’s hematoxylin (Gills Formula #2, CS401–1D; ThermoFisher Scientific, Waltham, MA), and dehydrated with ethanol and Clear-Rite 3 (#6901; ThermoFisher Scientific, Waltham, MA) before mounting with non-aqueous VectaMount (H-5000; Vector Laboratories, Burlingame, California, USA).

Techniques: Control, Staining, Two Tailed Test, Activation Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: IPF Pathogenesis is Dependent upon TGFβ Induction of IGF-1

doi: 10.1096/fj.201901719RR

Figure Lengend Snippet: (A) Anchorage Independent Growth (AIG) of AKR-2B murine fibroblast cells stimulated with TGFβ or vehicle in the presence of increasing concentrations of OSI-906 or vehicle (0.1% DMSO) after 8 days. Left: Representative images indicate colonies per treatment condition (red selection marks represent colonies with diameter ≥ 50 μm; scale bars measure 5 mm), and insert depicts a representative colony or individual cell size (scale bars measure 100 μm). Right: AIG was quantified by the number of colonies present under each condition (n=3). (B) Left: Scratch assays were performed with AKR-2B murine fibroblasts treated with vehicle (+V) or TGFβ (+β) and increasing concentrations of OSI-906 or vehicle. Red outlines indicate the leading edges of the wound. Right: The wound-healing response was quantified by the percent relative wound width measured at 0 and 24 hours (n=3). (C) Effect of increasing OSI-906 concentrations or vehicle on TGFβ-stimulated AKR-2B morphological transformation from a fibroblast morphology to differentiated myofibroblast phenotype (10x magnification) at 24 hours (n=3). (D) Stress fiber formation (F-actin polymerization associated with myofibroblast morphology) of confluent AKR-2B fibroblasts grown on glass coverslips and treated as in B (40x magnification). Filamentous actin (F-actin) is stained with phalloidin (red) and nuclei with DAPI (blue) (n=3). (E) Proliferative capacity of AKR-2B cells analyzed under normal (10% FBS) and stressful (0.1% FBS) growth conditions with increasing OSI-906 concentrations using BrdU incorporation (n=3). Data are presented as means −/+ Standard Error of the Mean (SEM) for the number of biological replicates indicated (n). Statistical significance was determined after computing single factor ANOVA and/or unpaired two-tailed Student’s t-test (p<0.05 (*), p<0.01 (**), p<0.005 (***), p<0.001 (****). Results demonstrate that inhibition of IGF-1R kinase activity with OSI-906 in AKR-2B murine fibroblasts effectively prevents TGFβ-dependent myofibroblast proliferation, differentiation, and profibrotic responses.

Article Snippet: Antigen specific primary antibodies to IGF-1 (4 μg/mL, AB9572; ABCAM, Cambridge, MA, USA) or p-IGF-1R (1:50 dilution, NB100–92555; Novus Biologicals, Centennial, Colorado, USA) were used by overnight 4°C incubation in a hydration container, counterstained with Mayer’s hematoxylin (Gills Formula #2, CS401–1D; ThermoFisher Scientific, Waltham, MA), and dehydrated with ethanol and Clear-Rite 3 (#6901; ThermoFisher Scientific, Waltham, MA) before mounting with non-aqueous VectaMount (H-5000; Vector Laboratories, Burlingame, California, USA).

Techniques: Selection, Transformation Assay, Staining, BrdU Incorporation Assay, Two Tailed Test, Inhibition, Activity Assay

Journal: FASEB journal : official publication of the Federation of American Societies for Experimental Biology

Article Title: IPF Pathogenesis is Dependent upon TGFβ Induction of IGF-1

doi: 10.1096/fj.201901719RR

Figure Lengend Snippet: A schematic diagram of the molecular mechanisms required for TGFβ-dependent stimulation of IGF-1 and the TGFβ-initiated profibrotic cellular responses mediated by IGF-1R activation.

Article Snippet: Antigen specific primary antibodies to IGF-1 (4 μg/mL, AB9572; ABCAM, Cambridge, MA, USA) or p-IGF-1R (1:50 dilution, NB100–92555; Novus Biologicals, Centennial, Colorado, USA) were used by overnight 4°C incubation in a hydration container, counterstained with Mayer’s hematoxylin (Gills Formula #2, CS401–1D; ThermoFisher Scientific, Waltham, MA), and dehydrated with ethanol and Clear-Rite 3 (#6901; ThermoFisher Scientific, Waltham, MA) before mounting with non-aqueous VectaMount (H-5000; Vector Laboratories, Burlingame, California, USA).

Techniques: Activation Assay