Journal: bioRxiv

Article Title: Prevention of Drug-Induced Lung Fibrosis via Inhibition of the MRTF/SRF Transcription Pathway

doi: 10.1101/2021.09.09.459118

Figure Lengend Snippet: Change in body weight during drug treatments. All animals received bleomycin treatment from day 1 -28 and test agents from days 1-42. Stress on the animals was observed in the rapid weight loss during the first week in the nintedanib and prednisolone groups, and a lack of significant weight gain over the 6 week study in all groups except 100 mg/kg CCG-257081. Due to excessive weight loss, the dose of prednisolone was lowered from 15 mg/kg to 5 mg/kg. Weight change time courses, which are significantly different from the vehicle control, are marked (*p < 0.05, **p < 0.01).

Article Snippet: Bleomycin (GoldBio, St. Louis, MO, #B-910-1G) was dissolved in sterile phosphate buffered saline (PBS, Gibco) at 3 mg/ml (15 mg/kg) and sterile filtered.

Techniques:

Journal: bioRxiv

Article Title: Prevention of Drug-Induced Lung Fibrosis via Inhibition of the MRTF/SRF Transcription Pathway

doi: 10.1101/2021.09.09.459118

Figure Lengend Snippet: CCG-257081 reduces inflammatory responses inherent to bleomycin-induced fibrotic disease. Histopathology scoring of A inflammation, B AT2 Hyperplasia and C fibrosis showed a trend in lower scores with higher concentrations of CCG-257081. Note: no data was recorded for the “no bleomycin” control group in AT2 Hyperplasia . The severity scores were: 0, no significant findings; 1, minimal; 2, mild; 3, moderate; 4, marked; 5, severe. * p < 0.05.

Article Snippet: Bleomycin (GoldBio, St. Louis, MO, #B-910-1G) was dissolved in sterile phosphate buffered saline (PBS, Gibco) at 3 mg/ml (15 mg/kg) and sterile filtered.

Techniques: Histopathology

Journal: bioRxiv

Article Title: Prevention of Drug-Induced Lung Fibrosis via Inhibition of the MRTF/SRF Transcription Pathway

doi: 10.1101/2021.09.09.459118

Figure Lengend Snippet: Light photomicrographs of lung tissue from mice in experimental groups: A Naïve control group, B Bleomycin + vehicle, C Bleomycin with low dose CCG-257081 (10 mg/kg), D Bleomycin with Prednisone treatment, E Bleomycin with medium dose CCG-257081 (30 mg/kg), F Bleomycin with Nintedanib treatment, and G Bleomycin with high dose CCG-257081 (100 mg/kg). Tissue sections histochemically stained with Masson’s Trichrome (blue: collagen, red: red blood cells, gray: alveolar space). Bleomycin-induced subpleural chronic alveolitis with increased collagen deposition (fibrosis; blue chromogen/stippled arrows) was observed, that was most severe in D (Prednisone treatment). No subpleural fibrotic lesions were noted with high dose CCG-257081 (G) or Nintedanib (F). p, pleural surface of lung; a, alveolar parenchyma; solid arrow, alveolar type II epithelial hyperplasia/hypertrophy.

Article Snippet: Bleomycin (GoldBio, St. Louis, MO, #B-910-1G) was dissolved in sterile phosphate buffered saline (PBS, Gibco) at 3 mg/ml (15 mg/kg) and sterile filtered.

Techniques: Staining

Journal: bioRxiv

Article Title: Prevention of Drug-Induced Lung Fibrosis via Inhibition of the MRTF/SRF Transcription Pathway

doi: 10.1101/2021.09.09.459118

Figure Lengend Snippet: Fibrotic markers were significantly decreased by MRTF/SRF inhibitors. A At 100 mg/kg CCG-257081, collagen content in the lungs was not significantly different from naive tissue (no bleomycin), while animals with prednisolone treatment had significantly greater amounts of collagen. B The pro-fibrotic biomarker, PAI-1, was significantly reduced in BALf by treatment with CCG-257081 at 100 mg/kg, but was not significantly reduced by any other treatment, including nintedanib or prednisolone. *p < 0.05, **p < 0.01.

Article Snippet: Bleomycin (GoldBio, St. Louis, MO, #B-910-1G) was dissolved in sterile phosphate buffered saline (PBS, Gibco) at 3 mg/ml (15 mg/kg) and sterile filtered.

Techniques: Biomarker Assay

Journal: Molecular Biology of the Cell

Article Title: Image-based drug screen identifies HDAC inhibitors as novel Golgi disruptors synergizing with JQ1

doi: 10.1091/mbc.E17-03-0176

Figure Lengend Snippet: Image-based chemical compound screen identifies HDAC inhibitors and DNA-damaging agents as novel Golgi-dispersing compounds. To identify novel compounds that modulate Golgi morphology, a screening platform was established. (A) Screening pipeline including cell seeding (A549 cells), treatment with compound library, staining for the cis -Golgi (GM130), the nucleus (Hoechst), the ER stress marker (GRP78), cytoplasm (Phalloidin), and image acquisition and processing. (B) Representative images of the negative control (vehicle-treated cells) as well as positive controls (BFA, doxorubicin, and nocodazole) and newly discovered Golgi-fragmenting drugs (Bleomycin, Vorinostat, 4-iodo-SAHA, Trichostatin A, Givinostat, and Pracinostat) are displayed. (C) Following image analysis, to exclude potential plate effects, the Golgi area of cells treated with the chemical library was normalized to the vehicle-treated sample present within the same plate. The corresponding survival ratios of treated cells are also shown. (D) Detailed view of compounds are shown, of which at least two of three replicates caused a ≥1.5-fold increase in Golgi area. The compound panel includes positive controls and novel Golgi-fragmenting compounds, which were selected for further investigation. *** p < 0.001 vs. #; see Materials and Methods .

Article Snippet: Compounds were obtained from the following companies: brefeldin A (Sigma-Aldrich), golgicide A (Santa Cruz Biotechnology), monensin (Enzo Life Sciences), AG-1478 (Sigma), tunicamycin (Santa Cruz Biotechnology), thapsigargin (Santa Cruz Biotechnology), nocodazole (Santa Cruz Biotechnology), (+)-JQ1 (Cayman Chemical), CBP30 (TargetMol), doxorubicin (Sigma), etoposide (Sigma), teniposide (Santa Cruz Biotechnology), mitomycin-C (Santa Cruz Biotechnology), cisplatin (Santa Cruz Biotechnology), hydroxyurea (Sigma), 5-fluorouracil (Sigma), gemcitabine (Santa Cruz Biotechnology), irinotecan (Santa Cruz Biotechnology), bleomycin (Santa Cruz Biotechnology), NU7441 (Selleckchem), KU55933 (Sigma), Flavopiridol (Santa Cruz Biotechnology), phorbol 12-myristate 13-acetate (PMA; Santa Cruz Biotechnology), Panobinostat (Selleckchem), Tubastatin (Selleckchem), Entinostat (Santa Cruz Biotechnology), Pracinostat (Selleckchem), Givinostat (Selleckchem), Triptolide (Santa Cruz Biotechnology), α-Amanitin (Santa Cruz Biotechnology), and Z-VAD-FMK (Santa Cruz Biotechnology).

Techniques: Drug discovery, Staining, Marker, Negative Control

Journal: Biosensors

Article Title: Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

doi: 10.3390/bios2030318

Figure Lengend Snippet: The cytotoxicity of bleomycin, mitomycin C and ethanol to flounder gill (FG) cells as determined by thiazolyl blue tetrazolium bromide ( MTT) assay. Data are expressed as mean ± SD.

Article Snippet: Bleomycin sulfate, Mitomycin C, Geneticin (G418), Lipofectamine LTX and PLUS reagents were purchased from Invitrogen, USA.

Techniques: MTT Assay

Journal: Biosensors

Article Title: Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

doi: 10.3390/bios2030318

Figure Lengend Snippet: Validation of the endogenous p53 -signaling pathway in FG cells. Examination of the responses of the transiently transformed FG cells to genotoxicant of bleomycin (30 μg/mL for 4 h) using firefly luciferase reporter plasmids of pGL 3 -p21-luc and pGL 3 -p53-luc and the Renilla luciferase internal reference plasmid of pRL-CMV. The intact FG cells (not transformed) were used as control. Data are expressed as mean ± SD.

Article Snippet: Bleomycin sulfate, Mitomycin C, Geneticin (G418), Lipofectamine LTX and PLUS reagents were purchased from Invitrogen, USA.

Techniques: Biomarker Discovery, Transformation Assay, Luciferase, Plasmid Preparation, Control

Journal: Biosensors

Article Title: Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

doi: 10.3390/bios2030318

Figure Lengend Snippet: The time-course responses of the stable p21FGLuc cells to model genotoxic and non-genotoxic agents. The stable p21FGLuc cells were exposed to 30 μg /mL bleomycin, 10 μg /mL mitomycin C and 30% (v/v) ethanol, respectively. The same stable p21FGLuc cells, not exposed to any toxicants but with the same volume of PBS, were used as control. ** Shows the highly significant difference (p < 0.01). Data are expressed as mean ± SD.

Article Snippet: Bleomycin sulfate, Mitomycin C, Geneticin (G418), Lipofectamine LTX and PLUS reagents were purchased from Invitrogen, USA.

Techniques: Control

Journal: Biosensors

Article Title: Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

doi: 10.3390/bios2030318

Figure Lengend Snippet: The dose-dependent responses of the stable p21FGLuc cells to model genotoxic and non-genotoxic agents. The stable p21FGLuc cells were exposed to increasing concentrations of bleomycin, mytomycin C and ethanol. Data are expressed as mean ± SD.

Article Snippet: Bleomycin sulfate, Mitomycin C, Geneticin (G418), Lipofectamine LTX and PLUS reagents were purchased from Invitrogen, USA.

Techniques:

Journal: Biosensors

Article Title: Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

doi: 10.3390/bios2030318

Figure Lengend Snippet: Comparison of the DNA damage-induced responses of the stable p21FGLuc cells to bleomycin, mitomycin C and ethanol. The p21FGLuc cells were exposed to 30 μg/mL bleomycin for 4 h, 10 μg /mL mitomycin C for 2 h and 30% (v/v) ethanol for 1 h, respectively. ** Shows the highly significant difference (p < 0.01). *** Shows the very highly significant difference (p < 0.001). Data are expressed as mean ± SD.

Article Snippet: Bleomycin sulfate, Mitomycin C, Geneticin (G418), Lipofectamine LTX and PLUS reagents were purchased from Invitrogen, USA.

Techniques: Comparison

Journal: Biosensors

Article Title: Development of a Fish Cell Biosensor System for Genotoxicity Detection Based on DNA Damage-Induced Trans-Activation of p21 Gene Expression

doi: 10.3390/bios2030318

Figure Lengend Snippet: Summary of the genotoxicity detection results using the fish cell biosensor system (p21FGLuc).

Article Snippet: Bleomycin sulfate, Mitomycin C, Geneticin (G418), Lipofectamine LTX and PLUS reagents were purchased from Invitrogen, USA.

Techniques: Concentration Assay

Journal: Mutagenesis

Article Title: Functional analysis of the new barley gene HvKu80 indicates that it plays a key role in double-strand DNA break repair and telomere length regulation.

doi: 10.1093/mutage/gev033

Figure Lengend Snippet: Figure 4. Relative HvKu80 gene expression levels in the parent cultivar ‘Sebastian’ (A), mutant ku80.c (B) and mutant ku80.j (C) after the treatment with bleomycin (100 µg/ml) and in the untreated material were analysed at various time points. ‘C’: seedlings after 36 h of growth at 24°C, ‘0’ in the untreated material denotes 36-h-old seedlings of barley that were treated with sterile water for 2 h at 37°C, ‘0’ in the treated material denotes 36-hold seedlings of barley that were treated with bleomycin for 2 h at 37°C. The value of the relative expression level was normalised to the standard control (HvARF1 gene, accession no. AJ508228.2). The expression level (2−ΔΔCt) in the untreated material was calculated relative to the control roots (C). The expression level (2−ΔΔCt) in the treated material was calculated relative to the untreated material at a particular time point. #Statistically significant difference vs. the control (C) (P < 0.05); *Statistically significant difference compared to the untreated material at the corresponding time point (P < 0.05).

Article Snippet: After this time, the seeds were incubated at 24°C for 36 h. Thirty-six-hour-old barley seedlings were transferred to 100 μg/ ml solution of bleomycin A5 hydrochloride (LKT Laboratories) and incubated at 37°C for 2 h. Upon the mutagenic treatment the seedlings were washed three times in sterile water for 15 min.

Techniques: Gene Expression, Mutagenesis, Sterility, Expressing, Control

Journal: Mutagenesis

Article Title: Functional analysis of the new barley gene HvKu80 indicates that it plays a key role in double-strand DNA break repair and telomere length regulation.

doi: 10.1093/mutage/gev033

Figure Lengend Snippet: Figure 5. The number of DSBs that were induced by bleomycin. Ethidium bromide-stained agarose gel visualising the induction of DSBs by bleomycin treatment in barley genomic DNA (A), concentration-dependent increase in DSB number in roots of the cultivar ‘Sebastian’ (B), the number of DSBs that were induced by 100 µg/ml bleomycin in the roots of the parent cultivar ‘Sebastian’ and the mutants ku80 after the bleomycin treatment (C). The DSB frequency was calculated for whole roots of the analysed genotypes. ‘M’: marker size 1 kb, ‘C’: measurement of DSB number was done on DNA isolated from whole roots after 36 h of growth at 24°C, ‘0’-treated material: measurement of DSB number was done on DNA isolated from whole 36-h-old roots of barley that were treated with bleomycin for 2 h at 37°C, *Statistically significant difference in relation to the parent cultivar ‘Sebastian’ (P < 0.05) at corresponding time points, #Statistically significant difference in relation to 0 µg/ml concentration of bleomycin (P < 0.05).

Article Snippet: After this time, the seeds were incubated at 24°C for 36 h. Thirty-six-hour-old barley seedlings were transferred to 100 μg/ ml solution of bleomycin A5 hydrochloride (LKT Laboratories) and incubated at 37°C for 2 h. Upon the mutagenic treatment the seedlings were washed three times in sterile water for 15 min.

Techniques: Staining, Agarose Gel Electrophoresis, Concentration Assay, Marker, Isolation

Journal: Mutagenesis

Article Title: Functional analysis of the new barley gene HvKu80 indicates that it plays a key role in double-strand DNA break repair and telomere length regulation.

doi: 10.1093/mutage/gev033

Figure Lengend Snippet: Figure 6. Number of MCD nuclei after the treatment of the roots of the cultivar ‘Sebastian’ and the mutants ku80.c and ku80.j with bleomycin. ‘C’: seedlings after 36 h of growth at 24°C, ‘0’-treated material: 36-h-old barley seedlings that were treated with bleomycin for 2 h at 37°C; *Statistically significant difference in relation to the parent cultivar ‘Sebastian’ (P < 0.05) at corresponding time points.

Article Snippet: After this time, the seeds were incubated at 24°C for 36 h. Thirty-six-hour-old barley seedlings were transferred to 100 μg/ ml solution of bleomycin A5 hydrochloride (LKT Laboratories) and incubated at 37°C for 2 h. Upon the mutagenic treatment the seedlings were washed three times in sterile water for 15 min.

Techniques:

Journal: Mutagenesis

Article Title: Functional analysis of the new barley gene HvKu80 indicates that it plays a key role in double-strand DNA break repair and telomere length regulation.

doi: 10.1093/mutage/gev033

Figure Lengend Snippet: Figure 7. Reduced growth of seedlings from the cultivar ‘Sebastian’ and the mutants ku80.c and ku80.j after treatment with bleomycin (100 µg/ml). Representative image of seedlings from the cultivar ‘Sebastian’ and the mutants ku80.c and ku80.j after treatment with bleomycin (A). Reduction of roots and shoots length of cultivar ‘Sebastian’ and the mutants ku80.c and ku80.j after bleomycin treatment (B). Control; ‘C’: seedlings after 60 h of growth at 24°C; scale = 3 cm, *Statistically significant difference in relation to root or shoot in control conditions (P < 0.05).

Article Snippet: After this time, the seeds were incubated at 24°C for 36 h. Thirty-six-hour-old barley seedlings were transferred to 100 μg/ ml solution of bleomycin A5 hydrochloride (LKT Laboratories) and incubated at 37°C for 2 h. Upon the mutagenic treatment the seedlings were washed three times in sterile water for 15 min.

Techniques: Control

Journal: JCI Insight

Article Title: Fibulin-1c regulates transforming growth factor–β activation in pulmonary tissue fibrosis

doi: 10.1172/jci.insight.124529

Figure Lengend Snippet: (A) Fbln1c deposition in lung sections from the nonfibrotic area and fibrotic area in patients with IPF and from healthy lung controls stained using immunohistochemistry (left); scale bar: 200 μm. Fbln1c-stained areas were quantified with normalization to the total area (right, n = 7–8). A single bleomycin challenge was used to induce pulmonary fibrosis in WT and Fbln1c–/– mice. Controls were challenged with PBS. #P < 0.05, compared to nonfibrotic IPF. (B) Stained areas of total Fbln1 were quantified around small airways with normalization to the perimeter of the basement membrane (Pbm) (n = 6–8). (C) Fbln1 protein levels were assessed using immunoblot of whole lung tissues (left), and fold change of densitometry was normalized to β-actin and quantified (right, n = 8). (D) Twenty-eight days after bleomycin or PBS challenge, lung sections were assessed for protein of the 1c isoform, Fbln1c, around small airways using immunofluorescence (top); scale bar: 50 μm. (Insets show expanded images of indicated regions; scale bar: 15 μm.) Fbln1c-stained areas around airways were quantified with normalization to the Pbm (bottom, n = 8). (E) Fbln1c protein area in parenchyma was determined using immunofluorescence (left); scale bar: 50 μm. Fbln1c-stained areas were quantified with normalization to total area (right, n = 8). (F) Fbln1c protein levels were assessed in whole lungs using immunoblot (top), and fold change of densitometry was quantified with normalization to β-actin (bottom, n = 8). Statistical differences were determined with 2-tailed Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with human healthy lung controls or PBS-challenged mouse controls.

Article Snippet: Experimental pulmonary fibrosis was induced by administration of a single dose of bleomycin sulfate (0.05 U/mouse, MP Biomedical) as described previously ( 21 – 23 ).

Techniques: Staining, Immunohistochemistry, Western Blot, Immunofluorescence

Journal: JCI Insight

Article Title: Fibulin-1c regulates transforming growth factor–β activation in pulmonary tissue fibrosis

doi: 10.1172/jci.insight.124529

Figure Lengend Snippet: A single bleomycin challenge was used to induce pulmonary fibrosis in WT and Fbln1c–/– mice that were assessed 28 days later. Controls received PBS. (A) Fbln1c and (B) Fbln1d mRNA levels in whole lungs determined using quantitative real-time PCR (qRT-PCR) (n = 6–8). (C) Lung sections were stained with Verhoeff-Van Gieson stain (left, scale bar: 500 μm; insets show expanded images of indicated regions; scale bar: 50 μm) and areas of collagen around small airways quantified with normalization to the Pbm (right, n = 8). (D) Total collagen levels were assessed by measuring hydroxyproline (left) and soluble collagen (right) in the whole lung tissues (n = 8). (E) Type I collagen (Col1a1) mRNA levels were measured in whole lungs using qRT-PCR (n = 8). (F) Col1a1 protein levels were measured in whole lungs using immunoblot (left), and fold change was quantified with normalization to β-actin (right, n = 8). (G) Collagen fibers were detected by second harmonic generation (SHG) microscopy (left), and fiber areas were calculated by forward (FSHG)/backward (BSHG) SHG ratios (right, n = 4–6; scale bar: 100 μm). Lung function in terms of (H) tissue damping, (I) tissue elastance, and (J) lung compliance was measured using invasive plethysmography and the forced oscillation technique (n = 5–8). Statistical differences were determined with 1-way ANOVA followed by Bonferroni’s posttest. *P < 0.05, **P < 0.01, and ****P < 0.0001 compared with PBS-challenged WT or Fbln1c–/– controls. †P < 0.05, and ††P < 0.01 compared with bleomycin-challenged WT controls. NS, not significant.

Article Snippet: Experimental pulmonary fibrosis was induced by administration of a single dose of bleomycin sulfate (0.05 U/mouse, MP Biomedical) as described previously ( 21 – 23 ).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Western Blot, Microscopy

Journal: JCI Insight

Article Title: Fibulin-1c regulates transforming growth factor–β activation in pulmonary tissue fibrosis

doi: 10.1172/jci.insight.124529

Figure Lengend Snippet: A single bleomycin challenge was used to induce pulmonary fibrosis in WT and Fbln1c–/– mice that were assessed 28 days later. Controls received PBS. (A) Fibronectin (Fn), (B) tenascin-C (Tnc), and (C) periostin (Postn) deposition in the basement membrane around small airways was assessed using immunohistochemistry and stained areas quantified with normalization to the Pbm (n = 24–40 airways from n = 4–8 mice per group). (D) Fn, (E) Tnc, and (F) Postn mRNA levels in lungs were determined using qRT-PCR (n = 6–8). (G) Fn, Tnc (variants 1 and 2), and Postn protein levels in whole lung tissues were assessed using immunoblot (top), and fold change was quantified using densitometry with normalization to β-actin (bottom, n = 5–8). Statistical differences were determined with 1-way ANOVA followed by Bonferroni’s posttest. *P < 0.05, and **P < 0.01 compared with PBS-challenged WT controls. †P < 0.05, ††P < 0.01, and ††††P < 0.0001 compared with bleomycin-challenged WT controls.

Article Snippet: Experimental pulmonary fibrosis was induced by administration of a single dose of bleomycin sulfate (0.05 U/mouse, MP Biomedical) as described previously ( 21 – 23 ).

Techniques: Immunohistochemistry, Staining, Quantitative RT-PCR, Western Blot

Journal: JCI Insight

Article Title: Fibulin-1c regulates transforming growth factor–β activation in pulmonary tissue fibrosis

doi: 10.1172/jci.insight.124529

Figure Lengend Snippet: A single bleomycin challenge was used to induce pulmonary fibrosis in WT and Fbln1c–/– mice that were assessed 28 days later. Controls received PBS. TGF-β (A) mRNA and (B) active protein levels in whole lung tissues measured using qRT-PCR and ELISA (n = 4–8). (C) LTBP1 levels in lungs measured using immunoblot (left) and fold change quantification using densitometry with normalization to β-actin (right, n = 5–8). (D) p-Smad3 protein levels in lungs measured using immunoblot (left) and fold change quantified using densitometry with normalization to vinculin (right, n = 5–8). (E) Immunoprecipitation (IP) of Fbln1c protein from whole lung tissues and detection of Fbln1c and LTBP1 binding using immunoblot (IB). IB analysis of lung tissues before (input) and after IP. Statistical differences were determined with 1-way ANOVA followed by Bonferroni’s posttest. *P < 0.05, and **P < 0.01 compared with PBS-challenged WT controls. †P < 0.05 compared with bleomycin-challenged WT controls.

Article Snippet: Experimental pulmonary fibrosis was induced by administration of a single dose of bleomycin sulfate (0.05 U/mouse, MP Biomedical) as described previously ( 21 – 23 ).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Immunoprecipitation, Binding Assay

Journal: JCI Insight

Article Title: Fibulin-1c regulates transforming growth factor–β activation in pulmonary tissue fibrosis

doi: 10.1172/jci.insight.124529

Figure Lengend Snippet: A single bleomycin challenge was used to induce pulmonary fibrosis in WT and Fbln1c–/– mice that were assessed 28 days later. Controls received PBS. (A) Tgfb mRNA levels in lungs were measured using qRT-PCR (n = 6). (B) LTBP1 levels in whole lung tissues were measured using immunoblot (left), and fold change was quantified using densitometry with normalization to β-actin (right, n = 6). Primary lung fibroblasts were isolated from whole lung tissues of naive WT and Fbln1c–/– mice and stimulated with TGF-β or control medium. (C) p-Smad3 protein levels in fibroblast lysates were measured using immunoblot (left), and fold change was quantified using densitometry with normalization to vinculin (right, n = 6). (D) Fibroblasts were stained with β-actin, and myofibroblasts were stained with α-SMA (left), and the percentage of myofibroblasts as a percentage of total fibroblasts was determined (right; scale bar: 500 μm; n = 6). (E) Col1a1 mRNA levels in fibroblast lysates were measured using qRT-PCR (n = 6). (F) Col1a1 protein levels in fibroblast lysates were measured using immunoblot, and fold change was quantified using densitometry with normalization to β-actin (right, n = 6). Primary mouse lung fibroblasts from WT mice were incubated with bronchoalveolar lavage fluid (BALF, 20 μL each mouse, 120 μL total) from WT and Fbln1c–/– mice after 28 days of bleomycin challenge and PBS controls for 6 hours. (G) Col1a1 and p-Smad3 protein in fibroblast lysates were measured using immunoblot, and (H) fold change was quantified using densitometry with normalization to vinculin (right, n = 6). Statistical differences were determined with 1-way ANOVA followed by Bonferroni’s posttest. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 compared with WT fibroblast controls. †P < 0.05, and ††P < 0.01 compared with TGF-β–stimulated WT fibroblast controls.

Article Snippet: Experimental pulmonary fibrosis was induced by administration of a single dose of bleomycin sulfate (0.05 U/mouse, MP Biomedical) as described previously ( 21 – 23 ).

Techniques: Quantitative RT-PCR, Western Blot, Isolation, Staining, Incubation