Journal: Nutrients

Article Title: Fermented Lettuce Extract Containing Nitric Oxide Metabolites Attenuates Inflammatory Parameters in Model Mice and in Human Fibroblast-Like Synoviocytes

doi: 10.3390/nu15051106

Figure Lengend Snippet: FLE suppresses the TGF-β/Smad signaling pathway in TGF-β-stimulated MH7A cells. ( A ) Representative morphological images of MH7A cells treated with FLE. Cells ( n = 3) were exposed to various FLE concentrations (62.5, 125, 250, and 500 μg nitrite/mL) for 24, 48, and 72 h. Viability was measured using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. ( B ) 5-Bromo-2′-deoxyuridine (BrdU) was incorporated into MH7A cells after FLE treatment for 72 h ( n = 3). ( C ) Effect of FLE and TGF-β (10 ng/mL) on MH7A cell viability at 72 h ( n = 3). ( D ) Western blotting of phospho-Smad2/3 and phospho-Smad1/5/9 expression in MH7A cell lysates after 72 h treatment with TGF-β ( n = 2−3). ( E ) RT-PCR detection of ACTA1 , COL1A , MMP2 , and MMP9 mRNA in MH7A cells ( n = 3). ( F ) Representative microscopic images of results from transwell migration assays. Data are shown as means ± SD ( n = 3). * p < 0.05, compared to control (CTL). † p < 0.05, compared to TGF-β. The plus (+) sign represents a combination of FLE and TGF-β, as in +62.5, +125, +250, and +500. The minus (−) sign represents untreated samples.

Article Snippet: Membranes were incubated overnight at 4 °C with primary antibodies phospho-Smad2/3 (1:1000, Cell Signaling, Danvers, MA, USA), phospho-Smad1/5/9 (1:1000, Cell Signaling), LC3B (1:1000; Cell Signaling), p62 (1:1000; Cell Signaling) and Beclin-1 (1:1000; Cell Signaling).

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Migration

Journal: International Journal of Medical Sciences

Article Title: Blockade of SK4 channels suppresses atrial fibrillation by attenuating atrial fibrosis in canines with prolonged atrial pacing

doi: 10.7150/ijms.69626

Figure Lengend Snippet: Effects of SK4 on the expression of CTGF, TGF-β1, Smad2, p-Smad2, Smad3 and p-Smad3 in atria . (A) Representative Western blot images of CTGF, TGF-β1, Smad2, p-Smad2, Smad3 and p-Smad3 expression levels in the atria. (B-G) The mean levels of CTGF, TGF-β1, Smad2, p-Smad2, Smad3 and p-Smad3 in the atria. *** P<0.001 versus the sham group. ### P<0.001 versus the pacing group. ▲▲ P<0.01 versus the sham group. ▲▲▲ P<0.001 versus the sham group. The data are presented as the mean ± SD, n=6.

Article Snippet: The membranes containing the sample proteins were incubated overnight with the primary antibodies against SK4 (Bioss, USA), collagen I, collagen III, matrix metalloproteinase (MMP)-2, MMP-9 (Abcam, USA), transforming growth factor β1 (TGF-β1) (Proteintech Group Inc., China), connective tissue growth factor (CTGF) (Proteintech Group Inc., China), Smad2 and phospho-specific Smad2 (p-Smad2), Smad3 and phospho-specific Smad3 (p-Smad3) , p38 and phospho-specific p38 (p-p38), ERK1/2 and phospho-specific ERK1/2 (p-ERK1/2) (Cell Signaling Technology, USA), c-Fos (Abcam, USA) and c-Jun (Abcam, USA) at 4 °C.

Techniques: Expressing, Western Blot

Journal: PLoS ONE

Article Title: TGF-Beta Induces Serous Borderline Ovarian Tumor Cell Invasion by Activating EMT but Triggers Apoptosis in Low-Grade Serous Ovarian Carcinoma Cells

doi: 10.1371/journal.pone.0042436

Figure Lengend Snippet: A, The endogenous Smad4 protein levels were analyzed in SBOT3.1 and MPSC1 cells using a western blot. B, SBOT3.1 and MPSC1 cells were treated with 10 ng/ml TGF-β for the indicated durations. The Smad2 and Smad3 phosphorylation levels were determined using western blot analysis with antibodies specific for the phosphorylated, activated forms of Smad2 (p-Smad2) and Smad3 (p-Smad3). The membranes were stripped and reprobed with antibodies directed against Smad2 and Smad3. C, SBOT3.1 and MPSC1 cells were treated with SB431542 (10 µM) in the presence or absence of 10 ng/ml TGF-β for 30 min. The Smad2 and Smad3 phosphorylation levels were analyzed by western blot.

Article Snippet: The monoclonal anti-phospho-Smad3, anti-Smad3, anti-Smad2, polyclonal anti-TGF-β receptor I, anti-TGF-β receptor II, anti-phospho-Smad2, anti-Smad4 and anti-caspase-3 antibodies were obtained from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot

Journal: BMC Complementary and Alternative Medicine

Article Title: Inhibition by curcumin of multiple sites of the transforming growth factor-beta1 signalling pathway ameliorates the progression of liver fibrosis induced by carbon tetrachloride in rats

doi: 10.1186/1472-6882-12-156

Figure Lengend Snippet: Primer sequences used in this study

Article Snippet: Antibodies included human polyclonal anti-TGF-β1 antibody (Promega, Madison, WI), rabbit anti-TGF-β1 polyclonal antibody (Boster Co., Ltd., Wuhan, China), goat polyclonal anti-CTGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit monoclonal anti-Smad2, anti-Smad3 and anti-Smad7 antibodies (Epitomics, Burlingame, CA), rabbit polyclonal anti-phospho-Smad2 ( Ser465/467 ) antibody (Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-collagen I antibody (Abcam, Cambridge, MA), rabbit polyclonal α-SMA antibody (Dako Diagnostic, San Antonio, TX), mouse monoclonal anti-GADPH antibody (Kangcheng Biotech Co., Ltd., Shanghai, China), and HRP-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin G (Beijing CowinBioscience Co., Ltd., Beijing, China).

Techniques:

Journal: BMC Complementary and Alternative Medicine

Article Title: Inhibition by curcumin of multiple sites of the transforming growth factor-beta1 signalling pathway ameliorates the progression of liver fibrosis induced by carbon tetrachloride in rats

doi: 10.1186/1472-6882-12-156

Figure Lengend Snippet: Effects of curcumin (Cur) on the expression of Smad2 and Smad3 proteins in rat liver tissue. ( A , B ) Immunohistochemical staining for Smad2 and Smad3 in the liver (original magnification, ×200). ( C , D ) Western blot analysis of hepatic Smad2 and Smad3 expression. Liver tissue was lysed and subjected to western blot analysis for Smad2 and Smad3 and the loading control GAPDH. Blots obtained from several experiments were analysed using densitometry, and the densitometric values were pooled from four animals per group and shown as means ± SD in the bar graph. * P < 0.05 for Cur/CCl 4 vs. PBS/CCl 4 , ** P < 0.01 for Cur/CCl 4 vs. PBS/CCl 4 .

Article Snippet: Antibodies included human polyclonal anti-TGF-β1 antibody (Promega, Madison, WI), rabbit anti-TGF-β1 polyclonal antibody (Boster Co., Ltd., Wuhan, China), goat polyclonal anti-CTGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit monoclonal anti-Smad2, anti-Smad3 and anti-Smad7 antibodies (Epitomics, Burlingame, CA), rabbit polyclonal anti-phospho-Smad2 ( Ser465/467 ) antibody (Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-collagen I antibody (Abcam, Cambridge, MA), rabbit polyclonal α-SMA antibody (Dako Diagnostic, San Antonio, TX), mouse monoclonal anti-GADPH antibody (Kangcheng Biotech Co., Ltd., Shanghai, China), and HRP-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin G (Beijing CowinBioscience Co., Ltd., Beijing, China).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot

Journal: BMC Complementary and Alternative Medicine

Article Title: Inhibition by curcumin of multiple sites of the transforming growth factor-beta1 signalling pathway ameliorates the progression of liver fibrosis induced by carbon tetrachloride in rats

doi: 10.1186/1472-6882-12-156

Figure Lengend Snippet: Curcumin induces Smad7 and inhibits Smad2, P-Smad2, and Smad3 mRNA and protein expression in liver tissue of CCl 4 injected rats. ( A - C ) The levels of Smad2 ( A ), Smad3 ( B ) and Smad7 ( C ) mRNA in liver tissue were determined by quantitative real-time RT-PCR. Smad2, Smad3 and Smad7 mRNA levels were normalized relative to GAPDH mRNA expression in each sample, and values are expressed as the mean ± SD fold increase over normal control group (n=6). *P < 0.01 for Cur/CCl 4 vs. PBS/CCl 4 . ( D - E ) Western blots analysis of hepatic P-Smad2 and Smad7 expression, with GAPDH as the loading control. Blots obtained from several experiments were analysed using densitometry, and the densitometric values were pooled from four animals per group and are shown as means ± SD in the bar graph. * P < 0.05 for Cur/CCl 4 vs. PBS/CCl 4 .

Article Snippet: Antibodies included human polyclonal anti-TGF-β1 antibody (Promega, Madison, WI), rabbit anti-TGF-β1 polyclonal antibody (Boster Co., Ltd., Wuhan, China), goat polyclonal anti-CTGF antibody (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit monoclonal anti-Smad2, anti-Smad3 and anti-Smad7 antibodies (Epitomics, Burlingame, CA), rabbit polyclonal anti-phospho-Smad2 ( Ser465/467 ) antibody (Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-collagen I antibody (Abcam, Cambridge, MA), rabbit polyclonal α-SMA antibody (Dako Diagnostic, San Antonio, TX), mouse monoclonal anti-GADPH antibody (Kangcheng Biotech Co., Ltd., Shanghai, China), and HRP-conjugated goat anti-rabbit and goat anti-mouse immunoglobulin G (Beijing CowinBioscience Co., Ltd., Beijing, China).

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

Journal: PLoS ONE

Article Title: NDRG2 Ameliorates Hepatic Fibrosis by Inhibiting the TGF-β1/Smad Pathway and Altering the MMP2/TIMP2 Ratio in Rats

doi: 10.1371/journal.pone.0027710

Figure Lengend Snippet: (A) Real-time PCR was used to detect the expression of Smad2, Smad3 and Smad7 in LX-2 cells with TGF-β1 and/or adenovirus treatment. (B) Western blotting was used to detect the expression of Smad2, phospho-Smad2, Smad3, phospho-Smad3 and Smad7 in LX-2 cells with TGF-β1 and/or adenovirus treatment. (C) Real-time PCR was used to detect the expression of MMP2 and TIMP2 in LX-2 cells following TGF-β1 and/or adenovirus treatment. β-actin was used as a loading control. All experiments were performed independently in triplicate. *P<0.05 compared with the TGF-β1 or TGF-β1+AdLacZ group.

Article Snippet: The blots were probed with the following primary antibodies: NDRG2 (Abnova), β-actin, Smad7 (Santa Cruz Biotechnology), α-SMA (Abcam), Smad2/phospho-Smad2 (pSmad2), Smad3/phospho-Smad3 (pSmad3) (Cell Signaling Technology), followed by incubation with species-matched secondary antibodies.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: PLoS ONE

Article Title: NDRG2 Ameliorates Hepatic Fibrosis by Inhibiting the TGF-β1/Smad Pathway and Altering the MMP2/TIMP2 Ratio in Rats

doi: 10.1371/journal.pone.0027710

Figure Lengend Snippet: (A) Real-time PCR was used to detect the expression of Smad2, Smad3 and Smad7 in rats with PBS or adenovirus treatment. (B) Western blotting was used to detect the expression of Smad2, phospho-Smad2, Smad3, phospho-Smad3 and Smad7 in rats with PBS or adenovirus treatment. (C) Real-time PCR was used to detect the expression of MMP2 and TIMP2 in rats following PBS or adenovirus treatment. β-actin was used as a loading control. “normal” means rats without treatment. *P<0.05 compared with the PBS or AdLacZ group.

Article Snippet: The blots were probed with the following primary antibodies: NDRG2 (Abnova), β-actin, Smad7 (Santa Cruz Biotechnology), α-SMA (Abcam), Smad2/phospho-Smad2 (pSmad2), Smad3/phospho-Smad3 (pSmad3) (Cell Signaling Technology), followed by incubation with species-matched secondary antibodies.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot

Journal: PLoS ONE

Article Title: NDRG2 Ameliorates Hepatic Fibrosis by Inhibiting the TGF-β1/Smad Pathway and Altering the MMP2/TIMP2 Ratio in Rats

doi: 10.1371/journal.pone.0027710

Figure Lengend Snippet: Primers for Real-time PCR.

Article Snippet: The blots were probed with the following primary antibodies: NDRG2 (Abnova), β-actin, Smad7 (Santa Cruz Biotechnology), α-SMA (Abcam), Smad2/phospho-Smad2 (pSmad2), Smad3/phospho-Smad3 (pSmad3) (Cell Signaling Technology), followed by incubation with species-matched secondary antibodies.

Techniques:

Journal: Nanotheranostics

Article Title: FGF2 engineered SPIONs attenuate tumor stroma and potentiate the effect of chemotherapy in 3D heterospheroidal model of pancreatic tumor

doi: 10.7150/ntno.38092

Figure Lengend Snippet: Effect of FGF2-SPION on the differentiation of hPSCs. Western blot (A) and quantitation showing the effect of FGF2 and FGF2-SPIONs at 250 ng/ml and 500 ng/ml on the protein expression of α-SMA (B), collagen-1 (col-1) (C) in hPSCs activated with 5 ng/ml TGF-β for 48 h compared to untreated hPSCs. Western blot and quantification showing the effect of FGF2 and FGF2-SPIONs on the phosphorylation of Smad2/3 (D) and ERK1/2 (E) in hPSCs activated with 5 ng/ml TGF-β for 1 h compared to untreated hPSCs. The protein expression levels for α-SMA and col-1 were normalized to β-actin, while pSmad2/3 and pERK1/2 were normalized to respective total protein levels. (F) Relative % growth of cells after 48 hours treatment with SPION, FGF2, or FGF2 SPION at concentration equal to 250 ng/ml or 500 ng/ml FGF2 and with or without TGF-β indicating no toxic effect exhibited by nanoparticles. (G) Representative immunofluorescence images showing the effect of FGF2 and FGF2-SPION on the protein expression of α-SMA and col-1 in TGF-β-activated hPSCs. Data represents mean + SEM for at least 3 independent experiments. Statistical differences are *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: Rabbit monoclonal anti-phospho-Smad2/3 , Cell Signaling , 1:1000 , .

Techniques: Western Blot, Quantitation Assay, Expressing, Concentration Assay, Immunofluorescence

Journal: Nanotheranostics

Article Title: FGF2 engineered SPIONs attenuate tumor stroma and potentiate the effect of chemotherapy in 3D heterospheroidal model of pancreatic tumor

doi: 10.7150/ntno.38092

Figure Lengend Snippet: Details of the antibodies used in the study.

Article Snippet: Rabbit monoclonal anti-phospho-Smad2/3 , Cell Signaling , 1:1000 , .

Techniques: