Journal: Journal of molecular and cellular cardiology

Article Title: Exogenous extracellular matrix proteins decrease cardiac fibroblast activation in stiffening microenvironment through CAPG

doi: 10.1016/j.yjmcc.2021.06.001

Figure Lengend Snippet: (A) Fibroblast CAPG expression was evaluated by western blot after 48h CAPG siRNA treatment. (B) CAPG expression was significantly decreased by siRNA treatment. (C) Fibroblast activation after CAPG knockdown was examined by immunostaining. (D) Decreasing CAPG expression significantly lowered the ratio of α-SMA positive fibroblast in TGF-β treated cells and control. (E) Collagen type I α1 expression was examined by western blot. (F) Knock down of CAPG level lowered collagen 1α1 expression in fibroblasts compared to control. (panel B, F: n=3 biological replicates, 2 technical replicates in each biological replicate, t-test, *p<0.05; panel D: n=3 biological replicates, 4 technical replicates in each biological replicate, one-way ANOVA and Tukey’s test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Data presented as mean ± standard deviation.)

Article Snippet: Membranes were blocked in 4% non-fat milk, followed by 2-hour incubation with 1:1000 diluted anti-LPXN, anti-CAPG, 1:60 diluted anti-collagen 1α1 (Developmental Studies Hybridoma Bank), and 1:20 000 diluted anti-GAPDH antibodies (Proteintech).

Techniques: Expressing, Western Blot, Activation Assay, Knockdown, Immunostaining, Control, Standard Deviation

Journal: Scientific reports

Article Title: Ketotifen directly modifies the fibrotic response of human skin fibroblasts.

doi: 10.1038/s41598-024-57776-7

Figure Lengend Snippet: Figure 1. αSMA gene expression and protein levels are reduced in TGFβ1-activated fibroblasts following ketotifen treatment. (A, B) HDFa and (C, D) WS1 fibroblasts were treated for 48 h with DMEM supplemented with 10% FBS as mock, 10 μM or 25 μM ketotifen, 10 ng/mL TGFβ1, or 10 ng/mL TGFβ1 with ketotifen added during the final 24 h. Gene expression of αSMA (ACTA2) was measured using RT-qPCR and normalized to housekeeping gene HPRT. HDFa cells were treated for 48 h under the same conditions and probed for αSMA protein by western blot. (E) A representative image of the blots is shown (left) and semi-quantitative assessments plotted (right) using GAPDH as loading control. Full-length blots are available in Supplementary Fig. 4. (F) COL1A1 was measured in HDFa cells treated with conditions using 25 μM ketotifen. Protein levels of pro-collagen 1α1 (G) and fibronectin (H) were determined by ELISA staining kits. Data shown as mean ± SEM. n = 3–6 per treatment condition. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. αSMA alpha-smooth muscle actin, GAPDH glyceraldehyde 3-phosphate dehydrogenase, HDFa human dermal fibroblasts (adult), SEM standard error of the mean, TGFβ1 transforming growth factor-beta.

Article Snippet: Antibodies used for protein concentration measurements were from pro-collagen 1α1 DuoSet ELISA (DY6220-05, R&D Systems) and fibronectin DuoSet ELISA (DY1918-05, R&D Systems) staining kits.

Techniques: Gene Expression, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Staining

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 1. SMOC2 is highly upregulated in mice and humans with kidney fibrosis. Quantitative immunostaining for SMOC2 (red) and αSMA (green) was performed on kidney sections obtained from mice at day 7 following (A) unilateral ureteral obstruction (UUO) or (B) folic acid injection (FA) (n = 5; 20× magnification). Scale bar: 50 μm. For the UUO model, contralateral kidney (CoK) tissue from day 14 was also included. Bottom panel in A shows magnified images taken at 40× from the selected regions of the middle row. Scale bar: 30 μm. Relative quantitation of SMOC2 and αSMA immunofluorescence, as represented in a box plot, was performed using representative images of 5 visual fields for each tissue analyzed. (C and D) Representative Western blot (n = 5/condition; Supplemental Figure 1, B and C [UUO and FA, respectively]) of SMOC2, αSMA, collagen 1α1, and fibronectin expression using kidney samples obtained from mice subjected to 7 and 14 days of UUO or FA. (E) Quantitative immunostaining for SMOC2 (red) and αSMA (green) in human kidneys with pathological fibrosis underlying chronic kidney disease (CKD) (n = 5) and nonfibrotic patients (n = 5; 20× magnification). Yellow scale bars: 50 μm. Bottom panel shows magnified images taken at 60× from the selected regions of the middle row. White scale bars: 25 μm. Relative quantitation of SMOC2 and αSMA immunofluorescence as represented in a box plot was performed using representative images of 5 visual fields for each tissue analyzed. (F) Urinary levels of SMOC2 and kidney injury molecule-1 (KIM-1) normalized to urinary creatinine were measured in patients with CKD (n = 13) compared with healthy volunteers (n = 13). Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 determined by t test. Yellow arrows, tubules. White arrows, interstitium.

Article Snippet: The following primary antibodies were used to detect the specific protein: anti-SMOC2 (1/250; Santa Cruz Biotechnology Inc., sc-67396), anti-αSMA (1/1,000; Sigma-Aldrich, A2547), anti–Collagen 1α1 (1:250; Novus, NB600-408), anti-Fibronectin (1:250; Abcam, ab23750), anti-GAPDH (1/5,000; Abcam, ab181602), anti–MLC2-P (Thr18/Ser19) (1/1,000; Cell Signaling Technology, 3674), anti–Pax-P (Tyr118) (1/1,000; Cell Signaling Technology, 2541), anti–Phospho-FAK (Tyr925) (1/1,000; Cell Signaling Technology, 3284), MYC (1/5000; Sigma-Aldrich, M4439) and Integrin β1 (1/1000; Santa Cruz, sc-8978).

Techniques: Immunostaining, Injection, Quantitation Assay, Immunofluorescence, Western Blot, Expressing

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 2. SMOC2-overexpressing mice are more susceptible to kidney fibrosis than WT mice. (A) Confirmation of SMOC2 overexpression in SMOC2 transgenic (SMOC2 Tg) mice by PCR (above, primers specific to recognize Tg insert) and Western blotting (below) (Supplemental Figure 3A). (B) Repre- sentative Western blot (n = 5/condition; Supplemental Figure 3B and Supplemental Figure 5B) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained from SMOC2 Tg and WT mice subjected to 7 and 14 days of unilateral ureteral obstruction (UUO). (C) Representative images of immunofluorescent staining for αSMA in CoK and fibrotic kidneys from WT and SMOC2 Tg mice at day 7 following UUO (n = 5/condition, 5 visual fields/ tissue). (D) Representative Western blot (n = 5/condition; Supplemental Figure 3C and Supplemental Figure 6B) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained from SMOC2 Tg and WT mice subjected to 7 and 14 days of folic acid (FA). (E) Representative images of immunofluorescent staining for αSMA of normal and fibrotic kidneys from WT and SMOC2 Tg mice at day 7 following FA (n = 5/condition, 10 visual fields/ tissue). (F) Representative images of picrosirius red (n = 5/condition, 10 visual fields/tissue) and Masson’s trichrome (n = 5/condition, 5 visual fields/ tissue) staining of CoK versus 7 and 14 day UUO–treated kidneys. (G) Representative images of picrosirius red and Masson’s trichrome staining of normal versus 7 and 14 day FA–treated kidneys (n = 5/condition, 5 visual fields/tissue). Confocal and light microscopy images are 20× magnification. Scale bars: 50 μM. Relative quantifications of images are represented as box plots, which describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 (CoK [UUO] or Normal [FA]) and #P < 0.05 (WT at respective time point) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: The following primary antibodies were used to detect the specific protein: anti-SMOC2 (1/250; Santa Cruz Biotechnology Inc., sc-67396), anti-αSMA (1/1,000; Sigma-Aldrich, A2547), anti–Collagen 1α1 (1:250; Novus, NB600-408), anti-Fibronectin (1:250; Abcam, ab23750), anti-GAPDH (1/5,000; Abcam, ab181602), anti–MLC2-P (Thr18/Ser19) (1/1,000; Cell Signaling Technology, 3674), anti–Pax-P (Tyr118) (1/1,000; Cell Signaling Technology, 2541), anti–Phospho-FAK (Tyr925) (1/1,000; Cell Signaling Technology, 3284), MYC (1/5000; Sigma-Aldrich, M4439) and Integrin β1 (1/1000; Santa Cruz, sc-8978).

Techniques: Over Expression, Transgenic Assay, Western Blot, Expressing, Staining, Light Microscopy

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 3. SMOC2 induces a fibroblast-to-myofibroblast transition. (A) RNAseq was performed using kidneys from SMOC2 Tg and WT mice at day 7 following UUO treatment. REVIGO treemap visualizations are shown for enriched gene ontology (GO) categories. Highly similar GO terms for cellular components are grouped and visualized by different colors and sizes of the rectangles using semantic similarity and enrichment P values. Representative Western blots of αSMA, collagen 1α1, and fibronectin from serum-deprived primary human kidney fibroblasts (B, n = 3/condition; Supplemental Figure 8C) and NIH3T3 fibroblasts (C, n = 3/condition; Supplemental Figure 8D) treated with 10 ng/ml SMOC2 with/without TGFβ1. (D) After 1 hour of antibody pretreatment, SMOC2 or TGFβ1 was treated to serum-deprived NIH3T3 cells for 24 hours and then tested for conventional fibrotic markers, while integrin β1 antibody was pretreated with NIH3T3 cells and then treated with SMOC2 (n = 3/condition; Supplemental Figure 8E). (E) NIH3T3 fibroblasts were transfected with SMOC2-MYC, empty vector control, or negative control MGP-MYC and then immunoprecipitated with a MYC (above) or integrin antibody (below). Western blots are representative immunoprecipitation experi- ments. (F) Representative Western blot for Phospho-Focal Adhesion Kinase (P-FAK) Y925, P-Myosin Light Chain (MLC) Ser19, and P-Paxillin Tyr118 from NIH3T3 cells treated with 10 ng/ml SMOC2 or 5 ng/ml TGFβ1 for 60 minutes (n = 5/condition; Supplemental Figure 8H). (G) Phalloidin staining of F-actin after NIH3T3 cells were treated 24 hours with 10 ng/ml SMOC2 or 5 ng/ml TGFβ1 (n = 3). Top row 40×, 25 μM. Bottom row 100×, 15 μM. Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 determined by t test.

Article Snippet: The following primary antibodies were used to detect the specific protein: anti-SMOC2 (1/250; Santa Cruz Biotechnology Inc., sc-67396), anti-αSMA (1/1,000; Sigma-Aldrich, A2547), anti–Collagen 1α1 (1:250; Novus, NB600-408), anti-Fibronectin (1:250; Abcam, ab23750), anti-GAPDH (1/5,000; Abcam, ab181602), anti–MLC2-P (Thr18/Ser19) (1/1,000; Cell Signaling Technology, 3674), anti–Pax-P (Tyr118) (1/1,000; Cell Signaling Technology, 2541), anti–Phospho-FAK (Tyr925) (1/1,000; Cell Signaling Technology, 3284), MYC (1/5000; Sigma-Aldrich, M4439) and Integrin β1 (1/1000; Santa Cruz, sc-8978).

Techniques: Western Blot, Transfection, Plasmid Preparation, Control, Negative Control, Immunoprecipitation, Staining

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 5. Genetic inhibition of SMOC2 limits folic acid–induced kidney fibrosis in mice. (A) Confirmation of SMOC2 deletion in SMOC2-KO mice by PCR (above, PCR primers specific to recognize knock-in insert) and Western blotting (below). (B) Representative Western blot (n = 4/group; Supplemental Fig- ure 11) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained at day 7 from SMOC2-KO and WT mice subjected to folic acid (FA) treatment. (C) Immunofluorescent αSMA staining of KO and WT kidneys at day 7 following with/out FA treatment (n = 4/group). (D) Masson’s trichrome staining of normal and FA-treated kidneys obtained at day 7 from KO and WT mice. Confocal and light microscopy images are 20× magnification. Scale bars: 50μM. Quantification of images is represented as box plots (n = 4/condition, 10 visual fields/mice), which describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 (WT normal) and #P <0.05 (WT at respective treatment) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: The following primary antibodies were used to detect the specific protein: anti-SMOC2 (1/250; Santa Cruz Biotechnology Inc., sc-67396), anti-αSMA (1/1,000; Sigma-Aldrich, A2547), anti–Collagen 1α1 (1:250; Novus, NB600-408), anti-Fibronectin (1:250; Abcam, ab23750), anti-GAPDH (1/5,000; Abcam, ab181602), anti–MLC2-P (Thr18/Ser19) (1/1,000; Cell Signaling Technology, 3674), anti–Pax-P (Tyr118) (1/1,000; Cell Signaling Technology, 2541), anti–Phospho-FAK (Tyr925) (1/1,000; Cell Signaling Technology, 3284), MYC (1/5000; Sigma-Aldrich, M4439) and Integrin β1 (1/1000; Santa Cruz, sc-8978).

Techniques: Inhibition, Knock-In, Western Blot, Expressing, Staining, Light Microscopy

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 6. Genetic inhibition of SMOC2 limits UUO-induced kidney fibrosis in mice. (A) Representative Western blot (n = 5/group; Supplemental Figure 12) of αSMA, collagen 1α1, fibronectin, and SMOC2 expression using kidney samples obtained at day 7 from SMOC2-KO and WT mice subjected to UUO. (B) Representative images (n = 3/group; 5 visual fields for each tissue analyzed) of immunofluorescent αSMA staining of KO and WT kidneys from normal mice and day 7 UUO mice. Relative quantitation is represented in a box plot as arbitrary units. (C) Masson’s trichrome staining of normal and 7-day UUO kidneys from WT and KO mice. Images of Masson’s trichrome staining are representative of 5–10 visual fields for each tissue analyzed. Quantification is represented in a box plot as arbitrary units (mice n = 5–6, 5–10 visual fields/mice). Confocal images are 20× magnification; scale bar: 50 μM. Light micros- copy images are 20× magnification; scale bar: 50μM. Box plots describe the median (line within box), upper and lower quartiles (bounds of box), and mini- mum and maximum values (bars). *P < 0.05 (WT CoK) and #P < 0.05 (WT at respective UUO) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: The following primary antibodies were used to detect the specific protein: anti-SMOC2 (1/250; Santa Cruz Biotechnology Inc., sc-67396), anti-αSMA (1/1,000; Sigma-Aldrich, A2547), anti–Collagen 1α1 (1:250; Novus, NB600-408), anti-Fibronectin (1:250; Abcam, ab23750), anti-GAPDH (1/5,000; Abcam, ab181602), anti–MLC2-P (Thr18/Ser19) (1/1,000; Cell Signaling Technology, 3674), anti–Pax-P (Tyr118) (1/1,000; Cell Signaling Technology, 2541), anti–Phospho-FAK (Tyr925) (1/1,000; Cell Signaling Technology, 3284), MYC (1/5000; Sigma-Aldrich, M4439) and Integrin β1 (1/1000; Santa Cruz, sc-8978).

Techniques: Inhibition, Western Blot, Expressing, Staining, Quantitation Assay

Journal: JCI insight

Article Title: Silencing SMOC2 ameliorates kidney fibrosis by inhibiting fibroblast to myofibroblast transformation.

doi: 10.1172/jci.insight.90299

Figure Lengend Snippet: Figure 7. Silencing SMOC2 reduces TGFβ1-induced fibrotic markers in vitro and folic acid–induced kidney fibrosis in mice. (A) Scheme of the exper- imental procedure for SMOC2 siRNA–transfected NIH3T3 cells. After 24 hours of treatment with SMOC2 siRNA or scrambled siRNA (ssiRNA), NIH3T3 fibroblasts were either treated with/without TGFβ1 for 24 hours. Representative Western blot (n = 3/condition; Supplemental Figure 14) was performed for SMOC2, αSMA, collagen 1α1, and fibronectin expression. (B) Scheme of the experimental procedure for SMOC2 siRNA– or ssiRNA-injected C57BL/6 mice treated with/out folic acid (FA). Mice were injected i.v. with 30 μg/200 μl of SMOC2 siRNA or ssiRNA 4 hours before and 2, 4, and 6 days after an i.p. injection of 250 mg/kg of FA. Representative Western blot (n = 5/group; Supplemental Figure 16) was performed for SMOC2, αSMA, collagen 1α1, and fibronectin. (C) Immunofluorescent αSMA staining of kidneys obtained from mice at day 7 following with/out FA either treated with ssiRNA or SMOC2 siRNA (n = 5). (D) Masson’s trichrome staining of normal and FA-treated kidneys obtained at day 7 following ssiRNA or SMOC2 siRNA administration. Confocal and light microscopy images are 20× magnification. Scale bars: 50 μM. Quantification of images is represented as a box plot (n = 5/condition, 10 visual fields/mice), which describe the median (line within box), upper and lower quartiles (bounds of box), and minimum and maximum values (bars). *P < 0.05 (ssiRNA + vehicle) and #P < 0.05 (ssiRNA respective treatment) determined by one-way ANOVA with Tukey post-hoc analysis.

Article Snippet: The following primary antibodies were used to detect the specific protein: anti-SMOC2 (1/250; Santa Cruz Biotechnology Inc., sc-67396), anti-αSMA (1/1,000; Sigma-Aldrich, A2547), anti–Collagen 1α1 (1:250; Novus, NB600-408), anti-Fibronectin (1:250; Abcam, ab23750), anti-GAPDH (1/5,000; Abcam, ab181602), anti–MLC2-P (Thr18/Ser19) (1/1,000; Cell Signaling Technology, 3674), anti–Pax-P (Tyr118) (1/1,000; Cell Signaling Technology, 2541), anti–Phospho-FAK (Tyr925) (1/1,000; Cell Signaling Technology, 3284), MYC (1/5000; Sigma-Aldrich, M4439) and Integrin β1 (1/1000; Santa Cruz, sc-8978).

Techniques: In Vitro, Transfection, Western Blot, Expressing, Injection, Staining, Light Microscopy

Journal: Aging Cell

Article Title: Vascular dysfunction in aged mice contributes to persistent lung fibrosis

doi: 10.1111/acel.13196

Figure Lengend Snippet: Delayed fibrosis resolution in aged mice following bleomycin challenge. (a) Young and aged mice were exposed to bleomycin and sacrificed after 30 and 75 days. Lungs were harvested and prepared for FACS sorting. (b) Col1a1 transcriptional analysis of FACS‐sorted GFP+/CD31−/CD45−/EpCAM− lung fibroblasts isolated from young and aged animals after bleomycin‐induced injury (young sham, N = 5; young 30 days, N = 8; young 75 days, N = 5; aged sham, N = 7; aged 30 days, N = 9; aged 75 days, N = 5). (c) Hydroxyproline assay was used to evaluate collagen deposition in the lungs (young sham, N = 8; young 14 days, N = 7; young 30 days, N = 9; young 75 days, N = 7). Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD , and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (d) Hydroxyproline assay was used to evaluate collagen deposition in the lungs (aged sham, N = 8; aged 14 days, N = 5; aged 30 days, N = 11; aged 75 days, N = 8). Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD , and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (e, f). Representative immunohistochemistry images and quantification of Collagen I by automated image analysis (young 75 days, N = 4; aged 75 days, N = 4). Data are non‐normally distributed, are expressed as median and IQR, and analyzed using non‐parametric Mann–Whitney test (* p < 0.05; ** p < 0.01).

Article Snippet: Mouse and human lung tissues were stained with Collagen 1α1 (dilution: 1:100; Novus biologicals, Centennial, CO, Cat# NB600‐408) or PECAM‐1 (dilution 1:2000; Abcam, Cat# ab28364) antibodies as described in Appendix .

Techniques: Isolation, Hydroxyproline Assay, Immunohistochemistry, MANN-WHITNEY

Journal: Aging Cell

Article Title: Vascular dysfunction in aged mice contributes to persistent lung fibrosis

doi: 10.1111/acel.13196

Figure Lengend Snippet: Vascular rarefaction accompanies persistent fibrosis in aged mice challenged with bleomycin. (a) Quantification of vascular density by automated image analysis (young sham, N = 4; young 30 days, N = 8; young 75 days, N = 9; aged sham, N = 4; aged 30 days, N = 6; aged 75 days, N = 11). Data passed Shapiro–Wilk normality test, are expressed as mean ± SD , and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (b) Representative IF images of mouse lung tissue stained with PECAM‐1 antibody. Scale bars: 100 μm. (c) Immunostaining of human tissue derived from normal or IPF lung for PECAM‐1 counterstained with hematoxylin. Magnifications: upper row, 4X, scale bars: 250 μm, lower row, 10X, scale bars: 100 μm. FF = fibroblastic foci. Arrows show areas occupied by microvessels in regions bordering FF. (d) Schematic for ex vivo lung tissue culture. Pieces of lungs from young and aged mice were embedded in collagen for 7 days in presence of 20 ng/ml VEGFA. (e) Collagen gel culture of lung explants derived from young and aged mice. (f) Vessel counts demonstrate reduction of sprouting outgrowth in aged mice. Data are non‐normally distributed, are expressed as median and IQR, and analyzed using non‐parametric Mann–Whitney test (* p < 0.05; *** p < 0.001).

Article Snippet: Mouse and human lung tissues were stained with Collagen 1α1 (dilution: 1:100; Novus biologicals, Centennial, CO, Cat# NB600‐408) or PECAM‐1 (dilution 1:2000; Abcam, Cat# ab28364) antibodies as described in Appendix .

Techniques: Staining, Immunostaining, Derivative Assay, Ex Vivo, MANN-WHITNEY

Journal: Aging Cell

Article Title: Vascular dysfunction in aged mice contributes to persistent lung fibrosis

doi: 10.1111/acel.13196

Figure Lengend Snippet: Loss of eNOS leads to sustained lung fibrosis in young animals following bleomycin challenge. (a) Nos3 transcriptional analysis of FACS‐sorted CD31+/GFP−/CD45−/EpCAM− lung ECs isolated from young and aged mice after bleomycin‐induced lung injury (young sham, N = 6; young 30 days, N = 10; young 75 days, N = 8; aged sham, N = 7; aged 30 days, N = 9; aged 75 days, N = 7). Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD , and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (b) Lung homogenates from WT and eNOS −/− mice were analyzed via Western blot using anti eNOS and anti GAPDH antibodies. (c) Hydroxyproline assay was used to evaluate collagen deposition in the lungs (WT sham, N = 7; WT 11 days, N = 3; WT 60 days, N = 10; eNOS −/− sham, N = 7; eNOS −/− 11 days, N = 3; eNOS −/− 60 days, N = 14). Data passed Shapiro–Wilk normality test, are expressed as mean ± SD , and analyzed using one‐way analysis of variance (followed by Tukey's post hoc test). (d) Masson's trichrome assay was used to stain lung tissue. (e) Transcriptional analysis of whole lung homogenates obtained from WT and eNOS −/− mice (WT 60 days, N = 7; eNOS −/− 60 days, N = 4). The reference group throughout all the genes analyzed in this panel is WT 60 days after bleomycin. Data passed Kolmogorov–Smirnov normality test, are expressed as mean ± SD , and analyzed using Student's t test (* p < 0.05; ** p < 0.01).

Article Snippet: Mouse and human lung tissues were stained with Collagen 1α1 (dilution: 1:100; Novus biologicals, Centennial, CO, Cat# NB600‐408) or PECAM‐1 (dilution 1:2000; Abcam, Cat# ab28364) antibodies as described in Appendix .

Techniques: Isolation, Western Blot, Hydroxyproline Assay, Staining

Journal: British Journal of Pharmacology

Article Title: Selective targeting of CREB‐binding protein/β‐catenin inhibits growth of and extracellular matrix remodelling by airway smooth muscle

doi: 10.1111/bph.13620

Figure Lengend Snippet: Primer sequences used

Article Snippet: The following antibodies were used: GAPDH (western blot 1:3000, mouse, Santa Cruz, #sc‐47724), phospho‐Rb (western blot 1:500, rabbit, Cell Signaling, #9308), α‐smooth muscle actin (α‐SMA; immunohistochemistry 1:100, Abcam, #ab5694), collagen 1α1 (western blot 1:1000, goat, SouthernBiotech, #1310‐01), β‐catenin (western blot 1:1000, immunofluorescence 1:300, mouse, BD Biosciences, #610154), non‐phospho (active) β‐catenin (western blot 1:500, immunofluorescence 1:300, rabbit, Cell Signaling, #8814), peroxidase‐conjugated anti‐mouse IgG (western blot 1:3000, rabbit, Sigma‐Aldrich, #A9044), peroxidase‐conjugated anti‐rabbit IgG (western blot 1:3000, goat, Sigma‐Aldrich, #A0545), peroxidase‐conjugated anti‐goat IgG (western blot 1:8000, rabbit, Sigma‐Aldrich, #A5420) and Alexa Fluor® 633‐conjugated anti‐rabbit IgG (immunofluorescence 1:500, goat, Invitrogen, #A‐21070).

Techniques: Sequencing

Journal: British Journal of Pharmacology

Article Title: Selective targeting of CREB‐binding protein/β‐catenin inhibits growth of and extracellular matrix remodelling by airway smooth muscle

doi: 10.1111/bph.13620

Figure Lengend Snippet: Impaired collagen 1α1 production following β‐catenin inhibition in vitro. (A) mRNA of airway smooth muscle (ASM) cells pre‐incubated with TGFβ1 (2 ng mL−1) for 24 h with or without ICG‐001, IQ‐1 or XAV‐939 (3 and 10 μM) was isolated and subjected to RT‐qPCR. Bars represent collagen 1α1 gene expression. Data represent five, eight and four independent experiments for ICG‐001, IQ‐1 and XAV‐939 treated samples respectively. (B) Collagen 1α1 immunoblot, normalized against GAPDH. Cells were treated with TGFβ1 (2 ng mL−1) for 48 h with or without ICG‐001, IQ‐1 or XAV‐939 (3 and 10 μM). Data represent five independent experiments. Data are expressed as the mean ± SEM. * Significant versus control, $ versus TGFβ1.

Article Snippet: The following antibodies were used: GAPDH (western blot 1:3000, mouse, Santa Cruz, #sc‐47724), phospho‐Rb (western blot 1:500, rabbit, Cell Signaling, #9308), α‐smooth muscle actin (α‐SMA; immunohistochemistry 1:100, Abcam, #ab5694), collagen 1α1 (western blot 1:1000, goat, SouthernBiotech, #1310‐01), β‐catenin (western blot 1:1000, immunofluorescence 1:300, mouse, BD Biosciences, #610154), non‐phospho (active) β‐catenin (western blot 1:500, immunofluorescence 1:300, rabbit, Cell Signaling, #8814), peroxidase‐conjugated anti‐mouse IgG (western blot 1:3000, rabbit, Sigma‐Aldrich, #A9044), peroxidase‐conjugated anti‐rabbit IgG (western blot 1:3000, goat, Sigma‐Aldrich, #A0545), peroxidase‐conjugated anti‐goat IgG (western blot 1:8000, rabbit, Sigma‐Aldrich, #A5420) and Alexa Fluor® 633‐conjugated anti‐rabbit IgG (immunofluorescence 1:500, goat, Invitrogen, #A‐21070).

Techniques: Inhibition, In Vitro, Incubation, Isolation, Quantitative RT-PCR, Expressing, Western Blot