Cell lines and Culture conditions
Human lung fibroblasts, IMR90, were obtained from the American Type Culture Collection (ATCC, #CCL-186) and cultured in EMEM media (ATCC, #30-2003) supplemented with 10% FBS (HyClone, #SH30109.03). Cells were maintained in a 37ºC humidified incubator buffered with 5% CO2. All experiments were performed at 70–80% cell densities.
Fibroblast differentiation conditions
IMR90 fibroblasts were differentiated to myofibroblasts with 5ng/mL Recombinant Human Transforming Growth Factor -β1 (TGF-β1) (Invitrogen, #PHG9204) for 48h in serum free media. TGF-β1 was reconstituted and stored according to manufacturer’s recommendations.
Antibodies and reagents: Antibodies for immunoblotting and immunocytochemistry: GFP (Abcam, #ab183734), VHL (Cell Signaling, #68547S), HIF1-⍺ (Cell Signaling Technology, #14179S), β Actin (Invitrogen, #PA5-59497), β Tubulin (Invitrogen, #MA5-16308), Fibronectin (Santa Cruz, #sc-59826), Collagen (Abcam, #ab34710), Smooth Muscle Actin (Abcam, #ab7817), Donkey Anti-Mouse Alexa Flour 568 (Invitrogen, #A10037), Donkey Anti- Rabbit Alexa Flour 568 (Invitrogen #A10042)
VH298 (Axon MedChem, #2810) was used at working concentrations of 100µM for 1h in serum media.
Adenovirus transduction
Ad-GFP (#1060), Ad-GFP #749 (amp), Ad-GFP#839 (amp) were purchased from Vector Biolabs at a viral titer of 4.5x1010PFU/mL, 1.2x1011PFU/mL, 1.1x1012 PFU/mL respectively. We obtained ~ 80% transduction for Ad-GFP 1060 (100MOI), Ad-GFP 749 (600MOI), and Ad-GFP 839 (600MOI) at 36h.
Protein extraction and immunoblotting
IMR90 cells were seeded on 6cm plates at a density of 163,000 cells. Protein extraction was performed at 4ºC using cold SDS lysis buffer containing protease and phosphatase inhibitors (1mM DTT, 1mM EDTA, 1µg/mL Leupeptin, 100µg/mL PMSF, and 1mM Sodium Orthovanadate). Proteins were separated by SDS-Poly acrylamide gel electrophoresis and immunoblotted for specific proteins of interest. β Actin and β Tubulin were used as loading controls. Quantification of immunoblots was performed using the Li-Cor Image Studio Software version 5.2. Pixel intensities of each protein were normalized to the loading control. Three independent experiments were averaged and fold differences between fibroblasts and myofibroblasts or myofibroblasts transduced with Ad-GFP 1060 and myofibroblasts transduced with adenoviruses Ad- GFP 739 or Ad-GFP 849 were plotted and shown as bar graphs. P values were determined using Student’s t-test.
Immunocytochemistry and imaging: For immunocytochemistry of FN, SMA, or COL, IMR90 cells were seeded on sterile coverslips in a 6-well plate at a density of 80,000 cells per well. The coverslips were fixed with 4% paraformaldehyde and permeabilized in 0.1% Triton X-100 for 1 min on ice. The cells were then blocked with 5% BSA-1x PBS for 1 h on ice. After blocking, FN antibody (1:70), SMA (1:1000) or COL (1:100) was added for 1h followed by 1h incubation with a secondary antibody. After secondary incubation, the cells were washed repeatedly with 0.2% Tween in 1xPBS. After washes, the cells were stained with the DNA stain DAPI (4, 6 diamidino-2-phenylindole dihydrochloride) (Roche, #1023627001) and mounded with Prolong gold anti-fade mound media (Invitrogen, #S36936) on glass slides. Images and z-stacks (1µM z-slice) were acquired using a Leica TCS SPEII confocal microscope at consistent acquisition parameters for each experiment.
Fibril count and quantification
Graphs in Fig. 2b, Fig. 2c, Fig. 3b, Fig. 4b were constructed by random fields of view within each sample being used to quantify the percentage of fibril containing cells. A FN or COL track of ~ 3µM in length was considered a fibril. A total of at least 25 cells were counted for each condition. Data in figures that include fibril counts are averages of 3 independent experiments. Student’s t-test was used to calculate P values.
SMA intensities
Graphs in Fig. 2d, Fig. 3c, Fig. 4c were constructed with the intensity plugin available in Image J. Cell containing random fields of view within each sample were used to quantify the SMA intensity values. Intensities shown in figures are averages of 3 independent experiments. Student’s t-test was used to calculate P values.
FUD and III-11C peptide treatment
FN fibril inhibitor experiments were performed by treating myofibroblasts with 500nM FUD (GenScript, #SC6004PF) or 500nM III-11C (GenScript, #SC6004PF) for 24h in serum media in addition to 5ng/mL TGF-β1. After peptide treatment, cells were fixed and processed for immunocytochemistry.
VHL Inhibitor treatment
VHL inhibitor experiments were performed by treating myofibroblasts with 100µM VH298 for 1h in serum media. Cells were then fixed and processed for immunocytochemistry or lysed in SDS lysis buffer for immunoblotting.
Statistical analysis
Immunoblots and immunocytochemistry experiments were performed as 3 independent trials and represented as averages ± SEM. Statistical significance was determined between fibroblasts and myofibroblasts or myofibroblasts transduced with Ad-GFP 1060 and myofibroblasts transduced with Ad-GFP 749 and Ad-GFP 839 for FN/COL. Statistical significance for fibrils counts, SMA intensities and immunoblot intensities were calculated using Student’s t-test. Statistical significance (P-value) was determined using GraphPad Prism 8.
Animal Models
All protocols concerning animal use were approved by the Institutional Animal Care and Use Committees at the Northern Arizona University and conducted in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Studies were conducted with 10–12 week old C57Bl/6 mice. Mice were housed in a temperature and humidity-controlled pathogen-free facility with a 12:12 hour light:dark cycle. Animals were housed within a limited access rodent facility and kept in groups of a maximum of 4 mice per cage. Mice were housed in polypropylene cages with solid bottoms and wood shavings or corn cobb as bedding material. At study termination, mice were euthanized via anesthesia overdose and exsanguination.
Bleomycin lung fibrosis model and viral transduction
IPF was established in C57Bl/6 mice (10–12 weeks) by oral aspiration (OA) of bleomycin (3.07 units/kg) on Day 0. Adenovirus (Ad-GFP-Empty, SOCS domain and SOCS domain mutant) was administered intranasally (IN) on Day 9. Animals were euthanized on Day 21 (5 animals in each group).
Collagen content (hydroxyproline assay) in the lung was determined from lung lysates collected on Day 21 of the study.
Immunohistochemistry and GFP-DAB staining and quantification
To determine optimum adenoviral titer for transduction, C57BL/6 mice were administered adenovirus 108 PFU and 109PFU or vehicle control (saline) on Day 0. On Day 3 following euthanasia, the lungs were inflated and fixed in formalin 10%. Tissues were processed in paraffin, sectioned and stained using GFP-DAB for assessment of transduction and H&E. Images were imported in HALO 3.0, regions of interest (lung tissue) annotated, and a quantitative image analysis algorithm performed. The algorithm (Cytonuclear V1.6) was used to segment nuclei based on hematoxylin staining, classify cells as either positive or negative for DAB staining and stratify positively stained cells as being 1+, 2 + or 3 + staining intensity (1 + being weak, 2 + being moderate, 3 + being strong staining intensity). To assign the H-score, a classification system was used to assess the total number of positive cells as well as the intensity of staining of each positive cell (on a scale of 0–3). The formula is calculated as [0*(ratio of negative cells) + 1*(ratio of 1 + cells) + 2*(ratio of 2 + cells) + 3*(ratio of 3 + cells)].