Journal: Experimental cell research

Article Title: Matrix metalloproteinase 9 inhibits the motility of highly aggressive HSC-3 oral squamous cell carcinoma cells.

doi: 10.1016/j.yexcr.2019.01.018

Figure Lengend Snippet: Fig. 4. (A) The amount of fibronectin in the control and shMMP9 HSC-3 cell homogenates (20 µg of soluble protein) was analysed with Western blotting. (B) Migration of the shMMP9 and control HSC-3 cells was analysed with a scratch wound assay on uncoated (empty) and 10 µg/ml fibronectin (Fn)-coated size 24 wells. The wounds were photographed with an EVOS photo microscope at 8 h and 24 h after scratching. The area of the open wound was measured with Fiji software and the results are presented as a percentage of wound closure (n = 4 scratch wounds analysed per condition). P-values were calculated using Student's T-test. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The membranes were blocked with 5% milk powder or 5% BSA (for Phospho-antibodies) in Tris-buffered saline 0.1% Tween 20 and incubated overnight with p44/42 MAPK (Erk1/2), Phospho-p44/ 42 MAPK (Erk1/2) (Thr202/Tyr204), Akt, Phospho-Akt (Ser473) (1:1000, all from Cell Signaling Technology), fibronectin (1:1000, ab24139) or beta Actin (1:2000) (both from Abcam) antibodies followed by biotinylated secondary antibodies (1:5000, DAKO) and Vectastain ABC kit (Vector Laboratories).

Techniques: Control, Western Blot, Migration, Scratch Wound Assay Assay, Microscopy, Software

Journal: Journal of diabetes & metabolism

Article Title: Thrombospondin-1 Deficiency Exacerbates the Pathogenesis of Diabetic Retinopathy

doi: 10.4172/2155-6156.S12-005

Figure Lengend Snippet: Increased GFAP and fibronectin expression in retinas from Akita/+; TSP1−/− mice. GFAP and fibronectin staining of the retinal sections from 7 month wild-type, Akita/+ and Akita/+; TSP1−/− male mice are shown (scale bar= 50 μM). Please note the increase in GFAP and fibronectin expression in Akita/+; TSP1−/− male mice compared to wild-type or Akita/+ male mice. Experiments were repeated with eyes from 5 mice with similar results.

Article Snippet: Sections were then incubated with rabbit anti-mouse fibronectin (1:500; Millipore), anti-GFAP (1:500; DAKO, Carpinteria, CA), anti-occludin (1:200; Zymed, South San Francisco CA) or mouse-anti-human TSP1 (1:500; Clone A6.1, Neo Marker, Fremont, CA) overnight at 4°C in humid environment.

Techniques: Expressing, Staining

Journal: Frontiers in Pharmacology

Article Title: Inhibition of Sirt2 Alleviates Fibroblasts Activation and Pulmonary Fibrosis via Smad2/3 Pathway

doi: 10.3389/fphar.2021.756131

Figure Lengend Snippet: Primers used in the real-time RT-PCR.

Article Snippet: Primary antibodies Sirt2 (1:1000; #9787), α -SMA (1:1000; #19245), Fibronectin (1:1000; #26836), phospho-Smad2 (1:1000; #3108), phospho-Smad3 (1:1000; #9520), Smad2/3 (1:1000; #8685), GAPDH (1:1000; #2118), β -actin (1:1000; #4970), and horseradish peroxidase-conjugated secondary antibody (1:5000; #7074) were all from Cell Signaling Technology.

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Inhibition of Sirt2 Alleviates Fibroblasts Activation and Pulmonary Fibrosis via Smad2/3 Pathway

doi: 10.3389/fphar.2021.756131

Figure Lengend Snippet: Sirt2 expression is increased in TGF- β 1 stimulated lung fibroblasts. (A,D) Expression of Fibronectin, α -SMA and Sirt2 proteins detected by Western blot in MRC-5 cells treated with 0 (control), 1, 2, 5, and 10 ng/ml TGF- β 1 for 24 h (B,E) Densitometric analyses of the Western blot in (A,D) . (C) Expression of Fibronectin and α -SMA mRNA detected by real-time RT-PCR in MRC-5 cells treated with 0 (control), 1, 2, 5, and 10 ng/ml TGF- β 1 for 24 h. (F) Expression of Sirt2 protein detected by Western blot in MRC-5 after 2 ng/ml TGF- β 1 exposure for 0, 3, 6, 12, 24, and 48 h. (G) Densitometric analyses of the Western blot in (F) . GAPDH was used as a loading control. The bars indicated mean ± SD of three separate experiments. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001 compared to the control.

Article Snippet: Primary antibodies Sirt2 (1:1000; #9787), α -SMA (1:1000; #19245), Fibronectin (1:1000; #26836), phospho-Smad2 (1:1000; #3108), phospho-Smad3 (1:1000; #9520), Smad2/3 (1:1000; #8685), GAPDH (1:1000; #2118), β -actin (1:1000; #4970), and horseradish peroxidase-conjugated secondary antibody (1:5000; #7074) were all from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Control, Quantitative RT-PCR

Journal: Frontiers in Pharmacology

Article Title: Inhibition of Sirt2 Alleviates Fibroblasts Activation and Pulmonary Fibrosis via Smad2/3 Pathway

doi: 10.3389/fphar.2021.756131

Figure Lengend Snippet: AGK2 decreases fibrogenic gene expression in TGF- β 1-induced lung fibroblasts activation. (A,C) Protein and mRNA expression of Sirt2, Fibronectin and α -SMA proteins by Western blot and real-time RT-PCR in MRC-5 cells treated with 2 ng/ml TGF- β 1 for 24 h and then added 10 μM AGK2 or DMSO for another 24 h in the presence of TGF- β 1. GAPDH was used as loading control. (B) Densitometric analyses of the Western blot in (A) . (D) Fibronectin and α -SMA was strongly expressed in response to TGF- β 1, and AGK2 decreased the expression by immunofluorescence staining. Green means Fibronectin and α -SMA staining; Blue means DAPI. The bars indicated mean ± SD of three separate experiments. DMSO, dimethyl sulfoxide; ** p <0.01, *** p <0.001, **** p <0.0001 compared to the TGF- β 1+DMSO group.

Article Snippet: Primary antibodies Sirt2 (1:1000; #9787), α -SMA (1:1000; #19245), Fibronectin (1:1000; #26836), phospho-Smad2 (1:1000; #3108), phospho-Smad3 (1:1000; #9520), Smad2/3 (1:1000; #8685), GAPDH (1:1000; #2118), β -actin (1:1000; #4970), and horseradish peroxidase-conjugated secondary antibody (1:5000; #7074) were all from Cell Signaling Technology.

Techniques: Gene Expression, Activation Assay, Expressing, Western Blot, Quantitative RT-PCR, Control, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Inhibition of Sirt2 Alleviates Fibroblasts Activation and Pulmonary Fibrosis via Smad2/3 Pathway

doi: 10.3389/fphar.2021.756131

Figure Lengend Snippet: Silencing Sirt2 inhibits fibrogenic gene expression in TGF- β 1-treated lung fibroblasts and IPF lung fibroblasts. (A,C) MRC-5 cells were transfected with NC siRNA or Sirt2 siRNA for 24 h, Sirt2 siRNA successfully downregulated Sirt2 protein and mRNA expression detected by Western blot and real-time RT-PCR. (B) Densitometric analyses of the Western blot in (A) . (D,F) Protein and mRNA expression of Sirt2, Fibronectin, and α -SMA by Western blot and real-time RT-PCR in MRC-5 cells transfected with NC siRNA or Sirt2 siRNA for 24 h in the absence or presence of 2 ng/ml TGF- β 1. (E) Densitometric analyses of the Western blot in (D) . (G) Expression of Sirt2, Fibronectin and α -SMA by Western blot in IPF lung fibroblasts treated with NC siRNA or Sirt2 siRNA for 24 h. (H) Densitometric analyses of the Western blot in (G) . GAPDH or β -actin was used as loading control. The bars indicated mean ± SD of three separate experiments. NC, negative control; siRNA, small interfering RNA. ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: Primary antibodies Sirt2 (1:1000; #9787), α -SMA (1:1000; #19245), Fibronectin (1:1000; #26836), phospho-Smad2 (1:1000; #3108), phospho-Smad3 (1:1000; #9520), Smad2/3 (1:1000; #8685), GAPDH (1:1000; #2118), β -actin (1:1000; #4970), and horseradish peroxidase-conjugated secondary antibody (1:5000; #7074) were all from Cell Signaling Technology.

Techniques: Gene Expression, Transfection, Expressing, Western Blot, Quantitative RT-PCR, Control, Negative Control, Small Interfering RNA

Journal: Frontiers in Pharmacology

Article Title: Inhibition of Sirt2 Alleviates Fibroblasts Activation and Pulmonary Fibrosis via Smad2/3 Pathway

doi: 10.3389/fphar.2021.756131

Figure Lengend Snippet: AGK2 attenuated bleomycin-induced pulmonary fibrosis and decreased the expreesion of p-Smad2/3 in vivo . (A) Representative image of IHC staining of Sirt2 (top, brown), Fibronectin (middle, brown) and α -SMA (bottom, brown) (magnification: ×400). (B) Quantitative analysis of IHC in (A) with Image-Pro Plus 6.0 software. (C,E) Protein expression of Sirt2, Fibronectin, α -SMA, p-Smad2/Smad2 and p-Smad3/Smad3 of lung tissues by Western blot. (D,F) Densitometric analyses of the Western blot in (C,E) . BLM, bleomycin. * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001.

Article Snippet: Primary antibodies Sirt2 (1:1000; #9787), α -SMA (1:1000; #19245), Fibronectin (1:1000; #26836), phospho-Smad2 (1:1000; #3108), phospho-Smad3 (1:1000; #9520), Smad2/3 (1:1000; #8685), GAPDH (1:1000; #2118), β -actin (1:1000; #4970), and horseradish peroxidase-conjugated secondary antibody (1:5000; #7074) were all from Cell Signaling Technology.

Techniques: In Vivo, Immunohistochemistry, Software, Expressing, Western Blot

Journal: Frontiers in Cell and Developmental Biology

Article Title: CCN2/CTGF—A Modulator of the Optic Nerve Head Astrocyte

doi: 10.3389/fcell.2022.864433

Figure Lengend Snippet: Immunoreactivity of fibronectin and labeling of filamentous actin in the ON and ONH in a murine glaucoma model. (A) Immunoreactivity of fibronectin (red, upper panel) is increased in the peripapillary sclera of βB1-CTGF1 mice compared to WT controls. Filamentous actin labeled by Phalloidin (red, lower panel) in increased in the ON of βB1-CTGF1 mice compared to WT controls. Nuclei are stained with Dapi (blue). (B) Quantification of immunohistochemical staining of fibronectin show a significant increase in the peripapillary sclera of TG animals compared to controls (TG: n = 4; WT: n = 5; ** p = 0.002). (C) Quantification of immunohistochemical staining of phalloidin labeled filamentous actin show a significant increase in the ON of TG animals compared to controls (TG: n = 5; WT: n = 5; *** p = 0.0001).

Article Snippet: After blocking with 1% BSA, 0.2% cold water fish skin gelatin (Sigma-Aldrich), 0.1% Triton-X-100 in 0.1 M phosphate buffer, frozen sections were incubated with rabbit anti-GFAP (1:1,000, Dako Denmark A/S,Glostrup, Denmark, Agilent Cat# Z0334, RRID:AB_1001338), rabbit anti-CTGF (1:400, Genetex Inc.,, Irvine, CA, United States, Cat# GTX26992, RRID:AB_369067), rabbit anti-fibronectin (1:500, Agilent Cat# A024502, RRID:AB_578510), chicken anti-GFAP (1:2000, LSBio (LifeSpan), Cat# LS-B4775-50, RRID:AB_10803257) or with phalloidin-rhodamin (1:500, 1 h at room temperature, Sigma-Aldrich) to label the actin cytoskeleton at 4°C overnight.

Techniques: Labeling, Staining, Immunohistochemical staining

Journal: Frontiers in Cell and Developmental Biology

Article Title: CCN2/CTGF—A Modulator of the Optic Nerve Head Astrocyte

doi: 10.3389/fcell.2022.864433

Figure Lengend Snippet: Analyses of ECM components in murine ON astrocytes following treatment with TGF-β2 and CCN2/CTGF. (A) Immunocytochemical staining against fibronectin (green, upper panel) showed a markedly increase following the treatment with 1 ng/ml TGF-β2 or 50 ng/ml CCN2/CTGF. Immunocytochemical staining against tropoelastin (green, lower panel) showed a pronounced increase after the treatment with 1 ng/ml TGF-β2 or 50 ng/ml CCN2/CTGF. Nuclei were stained with Dapi (blue). (B) Real-time RT-PCR analyses shows an intense upregulation of collagen 3α1 , tropoelastin and fibronectin mRNA in murine ON astrocytes after treatment with 1 ng/ml TGF-β2, 50 ng/ml CCN2/CTGF or 100 ng/ml CCN2/CTGF for 24 h compared to untreated control cells ( collagen 3α1 : n = 3; TGF-β2 *** p = 0.0000004, 50 ngCTGF ** p = 0.005, 100ngCTGF ** p = 0.008; fibronectin : n = 6, TGF-β2 * p = 0.00004, 50 ng CTGF * p = 0.016, 100 ngCTGF * p = 0.05; elastin : n = 5, TGF-β2 * p = 0.026, 50 ng CTGF *0.04, 100 ng CTGF * p = 0.03). mRNA expression was normalized to Gnb2l and mean value of control cells was set at 1. (C) Western Blot analysis show an increase in protein synthesis of collagen 3α1, elastin and fibronectin in murine ON astrocytes after treatment with 1 ng/ml TGF-β2, 50 ng/ml CCN2/CTGF or 100 ng/ml CCN2/CTGF for 24 h compared to untreated control cells (collagen 3α1: n = 3, TGF-β2 * p = 0.03, 100 ngCTGF ** p = 0.009; fibronectin: n = 5, ** p = 0.005, 50 ngCTGF ** p = 0.006, 100 ng CTGF * p = 0.02; tropoelastin: n = 5, TGF-β2 *** p = 0.0004, 50 ngCTGF ** p = 0.002, 100 ngCTGF *** p = 0.00001). GAPDH and α-tubulin were used to normalize protein synthesis and mean value of control cells was set at 1. Right panel shows representative Western Blots for all three proteins. Data represented as mean ± SD.

Article Snippet: After blocking with 1% BSA, 0.2% cold water fish skin gelatin (Sigma-Aldrich), 0.1% Triton-X-100 in 0.1 M phosphate buffer, frozen sections were incubated with rabbit anti-GFAP (1:1,000, Dako Denmark A/S,Glostrup, Denmark, Agilent Cat# Z0334, RRID:AB_1001338), rabbit anti-CTGF (1:400, Genetex Inc.,, Irvine, CA, United States, Cat# GTX26992, RRID:AB_369067), rabbit anti-fibronectin (1:500, Agilent Cat# A024502, RRID:AB_578510), chicken anti-GFAP (1:2000, LSBio (LifeSpan), Cat# LS-B4775-50, RRID:AB_10803257) or with phalloidin-rhodamin (1:500, 1 h at room temperature, Sigma-Aldrich) to label the actin cytoskeleton at 4°C overnight.

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