SOCS domain targets ECM assembly in lung fibroblasts and experimental lung fibrosis

Figures

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  Fig. 1. SOCS domain degrades VHL protein in myofibroblasts. (A,B,D) IMR90 lung fibroblasts were untreated or differentiated with 5 ng/mL TGFβ for 48 h. Differentiated myofibroblasts were then transduced with Ad-Empty 100 MOI, Ad-Domain 600 MOI or Ad-Mutant 600 MOI for ~ 36 h. Cells were lysed in SDS lysis buffer and immunoblotted for SOCS-GFP fusion protein, VHL and HIF1-⍺. β-Actin and β-Tubulin were used as the loading controls. (C,E) Quantification of VHL and HIF1-⍺ normalized to their respective loading controls, on immunoblots are represented as averages of three independent trials. Statistical significance between fibroblasts and myofibroblasts and Ad-Empty and Ad-Domain or Ad-Mutant P-values were determined using Student’s t-test.

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  Fig. 2. SOCS domain fragments FN matrix and reduces levels of SMA protein in myofibroblasts. (A) IMR90 lung fibroblasts were untreated or differentiated with 5 ng/mL TGFβ for 48 h. Differentiated myofibroblasts were then transduced with Ad-Empty (100 MOI), Ad-Domain (600 MOI) or Ad-Mutant (600 MOI) for ~ 36 h and immunostained for FN (red), COL (red) or SMA (red) and nuclear stain DAPI (blue). Myofibroblasts transduced with Ad-Empty (100 MOI), Ad-Domain (600 MOI) or Ad-Mutant (600 MOI) appear green (GFP reporter). Scale bar = 10 µm. (B,C) GFP reporter-expressing transduced cells as in (A) were quantified for percent of fibronectin fibril- containing cells and collagen fibril-containing cells respectively. Bar graphs are averages of three independent experiments. Student t-test was used to calculate P values. (D) Transduced cells expressing the GFP reporter as in (A) were used to quantify the SMA intensity values. Intensities shown in the graph are averages of three independent experiments. Student’s t-test was used to calculate P values.

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  Fig. 3. SOCS domain reduces differentiation of fibroblasts to myofibroblasts. (A) IMR90 lung fibroblasts were untreated or transduced with Ad-Empty (100 MOI), Ad-Domain (600 MOI) or Ad-Mutant (600 MOI) for ~ 36 h. Cells were then differentiated with 5 ng/mL TGFβ for 48 h. Fibroblasts, myofibroblasts (untransduced) and transduced myofibroblasts were immunostained for FN (red) or SMA (red) and nuclear stain DAPI (blue). Myofibroblasts transduced with Ad-Empty (100 MOI), Ad-Domain (600 MOI) or Ad-Mutant (600 MOI) appear green (GFP reporter). Scale bar = 10 µm. (B) Transduced cells as in (A) were quantified for percent of fibronectin fibril- containing cells and represented as bar graphs. Only cells that expressed the GFP reporter were included in the analysis. (C) Transduced cells expressing the GFP reporter were used to quantify the SMA intensity values. Intensities shown in the graph are averages of three independent experiments. Student’s t-test was used to calculate P values.

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  Fig. 4. FN fibril inhibition does not decrease SMA intensity in myofibroblasts. (A) IMR90 lung fibroblasts were untreated or differentiated with 5 ng/mL TGFβ. Differentiated myofibroblasts were then treated with 500 nM FUD or 500 nM IIIC-11C for 24 h in addition to 5 ng/mL TGFβ. Cells were immunostained for FN (red) or SMA (red) and nuclear stain, DAPI (blue). Scale bar = 10 µm. (B) Myofibroblasts and myofibroblasts treated with FUD or IIIC were quantified for fibronectin fibril- containing cells or (C) used to quantify the SMA intensity values. Bar graphs are averages of three independent trials. Student’s t-test was used to determine P values.

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  Fig. 5. VHL protein inhibition does not decrease SMA intensity in myofibroblasts. (A) IMR90 lung fibroblasts were untreated or differentiated with 5 ng/mL TGFβ for 48 h. Differentiated myofibroblasts were then treated with 100 µM VH298 for 1 h. Cells were then lysed in SDS buffer and immunoblotted for HIF1-⍺. β-Tubulin was used as the loading control. (B) Fibroblasts, myofibroblasts and myofibroblasts treated as in (A) were immunostained for FN (red) or SMA (red) and nuclear stain, DAPI (blue). Scale bar = 10 µm.

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  Fig. 6. Intranasal delivery of Adenoviral-GFP is localized to lung tissue and SOCS domain delivery significantly reduces collagen deposition in fibrotic lung. (A) C57BL/6 mice were intranasally administered vehicle (phosphate buffered saline) and adenoviral-GFP on Day 0 at 108 PFU and 109 PFU as depicted. Lung tissue was collected on Day 5 and Formalin Fixed paraffin embedded (FFPE) lung tissue was subjected to immunohistochemistry (IHC) staining using GFP-DAB antibody. Increased transduction of Ad-GFP at 109 PFU was observed compared to transduction at 108 PFU 5 days following intranasal delivery of Ad-GFP and vehicle control (n = 3). Magnification = 20x. Scale bar = 10 μm. (B) FFPE lung tissue sections analyzed for GFP expression. Top row depicts GFP-DAB staining and bottom row shows nuclei (blue). (C) Positive stain intensity from the bottom row in FIG. 6B, assigned using the scores: 1 + yellow, 2 + orange and 3 + red was used to quantify extent and intensity of GFP represented using the histoscore (H-score). Bar graph represents mean ± SD, n = 3. Statistical significance between vehicle (phosphate buffered saline) versus transduced and P-values (*P < 0.05, ***P < 0.001) are depicted.…