Journal: Tissue Engineering. Part A

Article Title: Response of Fibroblasts to Transforming Growth Factor-?1 on Two-Dimensional and in Three-Dimensional Hyaluronan Hydrogels

doi: 10.1089/ten.tea.2012.0094

Figure Lengend Snippet: Proliferation effect of transforming growth factor–β1 (TGF-β1) on cells plated on tissue culture polystyrene (TCP) (A), on Carbylan-GSX (B), and in three-dimensional (3D) Carbylan-GSX (C) at 2000 cells/well of a 96-well plate. After 24 h starvation, cells were treated with various doses (0.1 to 20 ng/mL) of TGF-β1 for 72 h. Cell numbers in quadruplicate were monitored by ATP levels (RLU). **p<0.01.

Article Snippet: Antibodies included hPH (collagen biosynthase; Millipore, Billerica, MA), α-SMA (myofibroblast marker; Sigma), type I and type II TGF-β1 receptor (TGFBRI and TGFBRII; Abcam, Cambridge, MA).

Techniques:

Journal: Tissue Engineering. Part A

Article Title: Response of Fibroblasts to Transforming Growth Factor-?1 on Two-Dimensional and in Three-Dimensional Hyaluronan Hydrogels

doi: 10.1089/ten.tea.2012.0094

Figure Lengend Snippet: Representative images of human vocal fold fibroblasts (hVFF) on TCP, two-dimensional (2D) hyaluronan (HA), and in 3D HA in response to TGF-β1 by hPH/alpha smooth muscle actin (α-SMA) double-immunofluorescence analysis. Morphological analysis of hVFF cultured on the TCP surface without TGF-β1 showed a typical spindle shape (A) and on the surface of HA cells showed the similar shape (B). However, hVFF were of smaller rounded morphology in 3D HA (C). Double-immunostaining of the cells with 5 ng/mL TGF-β1 treatment demonstrated strong expression of α-SMA along cell axis on the TCP surface (D, red), low expression on HA (E) and in HA (F, red as arrow). Nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar: 60 μm.

Article Snippet: Antibodies included hPH (collagen biosynthase; Millipore, Billerica, MA), α-SMA (myofibroblast marker; Sigma), type I and type II TGF-β1 receptor (TGFBRI and TGFBRII; Abcam, Cambridge, MA).

Techniques: Immunofluorescence, Cell Culture, Double Immunostaining, Expressing, Staining

Journal: Tissue Engineering. Part A

Article Title: Response of Fibroblasts to Transforming Growth Factor-?1 on Two-Dimensional and in Three-Dimensional Hyaluronan Hydrogels

doi: 10.1089/ten.tea.2012.0094

Figure Lengend Snippet: Quantitative real-time polymerase chain reaction (qPCR) and Western blot analyses of α-SMA expression in the hVFF. Cells grown on TCP, 2D HA, and in 3D HA were treated with various doses of TGF-β1. After 24 h, α-SMA mRNA levels were analyzed by qPCR (A). Data are expressed as mRNA expression for the α-SMA gene (ng/μL) relative to β-Actin (ng/μL). Values represent mean±SD of triplicated assays. *p<0.05; **p<0.01. After 48 h of TGF-β1 treatment, quantification of α-SMA protein levels of whole-cell lysates were analyzed by Western blot analysis (B). The first lane (MW) was the protein ladder. GAPDH (37 kDa) was used to demonstrate equal loads of total protein amount in different lanes.

Article Snippet: Antibodies included hPH (collagen biosynthase; Millipore, Billerica, MA), α-SMA (myofibroblast marker; Sigma), type I and type II TGF-β1 receptor (TGFBRI and TGFBRII; Abcam, Cambridge, MA).

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

Journal: Tissue Engineering. Part A

Article Title: Response of Fibroblasts to Transforming Growth Factor-?1 on Two-Dimensional and in Three-Dimensional Hyaluronan Hydrogels

doi: 10.1089/ten.tea.2012.0094

Figure Lengend Snippet: Effect of TGF-β1 on the gene expression of ECM components. hVFF on TCP, 2D HA, and in 3D HA were treated with various doses of TGF-β1 for 24 h, and were subjected to qPCR. Data are expressed as mRNA expression for Collagen I (Col1), III (Col3), and fibronectin (FN) gene (ng/μL) relative to β-Actin (ng/μL). Values represent mean±SD of triplicated assays. *p<0.05; **p<0.01. (A) TGF-β1-inducible Col1 mRNA levels. (B) TGF-β1-inducible Col3 mRNA levels. (C) TGF-β1-inducible FN mRNA levels.

Article Snippet: Antibodies included hPH (collagen biosynthase; Millipore, Billerica, MA), α-SMA (myofibroblast marker; Sigma), type I and type II TGF-β1 receptor (TGFBRI and TGFBRII; Abcam, Cambridge, MA).

Techniques: Expressing

Journal: Tissue Engineering. Part A

Article Title: Response of Fibroblasts to Transforming Growth Factor-?1 on Two-Dimensional and in Three-Dimensional Hyaluronan Hydrogels

doi: 10.1089/ten.tea.2012.0094

Figure Lengend Snippet: Regulation of matrix metalloproteinase (MMP1 and MMP2) activity by culture condition. hVFF grown on TCP, 2D HA, and 3D HA were starved for 24 h before the addition of various doses of TGF-β1 for 48 h. Equal amounts of cell supernatant were analyzed by the Zymogram assay. (A) MMP1 activity assay was performed by casein zymogram. (B) MMP2 activities were assayed by gelatin zymogram.

Article Snippet: Antibodies included hPH (collagen biosynthase; Millipore, Billerica, MA), α-SMA (myofibroblast marker; Sigma), type I and type II TGF-β1 receptor (TGFBRI and TGFBRII; Abcam, Cambridge, MA).

Techniques: Activity Assay

Journal: Tissue Engineering. Part A

Article Title: Response of Fibroblasts to Transforming Growth Factor-?1 on Two-Dimensional and in Three-Dimensional Hyaluronan Hydrogels

doi: 10.1089/ten.tea.2012.0094

Figure Lengend Snippet: Effect of TGF-β1 on MMP1, MMP2, and TIMP3 mRNA expression. hVFF seeded on TCP, 2D HA, and 3D HA were treated with various doses of TGF-β1 for 24 h, and were subjected to qPCR. Data are expressed as mRNA expression for MMP1, MMP2, and TIMP3 (ng/μL) relative to β-Actin (ng/μL). The ratios of MMPs to TIMP3 were calculated by β-Actin normalized MMPs over β-Actin normalized TIMP3. Values represent mean±SD of triplicated assays. **p<0.01. (A) TGF-β1-inducible MMP1 mRNA levels; (B) TGF-β1-inducible MMP2 mRNA levels; (C) TGF-β1-inducible TIMP3 mRNA levels; (D) ratios of MMP1 expression to TIMP3; (E) ratios of MMP2 expression to TIMP3.

Article Snippet: Antibodies included hPH (collagen biosynthase; Millipore, Billerica, MA), α-SMA (myofibroblast marker; Sigma), type I and type II TGF-β1 receptor (TGFBRI and TGFBRII; Abcam, Cambridge, MA).

Techniques: Expressing

Journal: Tissue Engineering. Part A

Article Title: Response of Fibroblasts to Transforming Growth Factor-?1 on Two-Dimensional and in Three-Dimensional Hyaluronan Hydrogels

doi: 10.1089/ten.tea.2012.0094

Figure Lengend Snippet: Effect of TGF-β1 on the mRNA expression of TGF-β receptors. hVFF seeded on TCP, 2D HA, and in 3D HA were treated with various doses of TGF-β1 for 24 h, and were subjected to qPCR. Data are expressed as mRNA expression for type I (TGFBRI) and type II (TGFBRII) of TGF-β receptor (ng/μL) relative to β-Actin (ng/μL). Values represent mean±SD of triplicated assays. **p<0.01. (A) TGF-β1-inducible TGFBRI mRNA levels. (B) TGF-β1-inducible TGFBRII mRNA levels.

Article Snippet: Antibodies included hPH (collagen biosynthase; Millipore, Billerica, MA), α-SMA (myofibroblast marker; Sigma), type I and type II TGF-β1 receptor (TGFBRI and TGFBRII; Abcam, Cambridge, MA).

Techniques: Expressing

Journal: Tissue Engineering. Part A

Article Title: Response of Fibroblasts to Transforming Growth Factor-?1 on Two-Dimensional and in Three-Dimensional Hyaluronan Hydrogels

doi: 10.1089/ten.tea.2012.0094

Figure Lengend Snippet: Expression of TGF-β receptors in hVFF were detected by immunocytochemistry staining. TGFBRI (A, red) and TGFBRII (B, green) were observed in the cells on TCP (a, d), 2D HA (b, e), and in 3D HA (c, f) conditions treated with either medium alone (a, b, c) or 5 ng/mL TGF-β1 (d, e, f) for 48 h. Nuclei were counter-stained with DAPI. Scale bar: 60 μm.

Article Snippet: Antibodies included hPH (collagen biosynthase; Millipore, Billerica, MA), α-SMA (myofibroblast marker; Sigma), type I and type II TGF-β1 receptor (TGFBRI and TGFBRII; Abcam, Cambridge, MA).

Techniques: Expressing, Immunocytochemistry, Staining

Journal: British Journal of Pharmacology

Article Title: Low‐concentration DMSO accelerates skin wound healing by Akt / mTOR ‐mediated cell proliferation and migration in diabetic mice

doi: 10.1111/bph.15052

Figure Lengend Snippet: DMSO promotes the migration of diabetic mouse‐derived primary keratinocytes in an indirect manner mediated by fibroblast‐derived TGF‐β1. (a) Migration of the keratinocytes by transwell assays after treatment with or without 5‐mM DMSO or TGF‐β1 for 24 h. Data are presented as the mean ± SEM with n = 5 for all groups, analysed by one‐way ANOVA with Kruskal–Wallis test for multiple comparison. Significance level: *P < 0.05, compared with the control group. NS, not significant. (b) Representative H&E‐stained sections of wound tissue 8 days after injury. The arrow indicates the length of the migrating epithelial tongue. Data are presented as the mean ± SEM with n = 6 for all groups, analysed by Student's t test. Significance level: *P < 0.05, 5‐mM DMSO versus the control group. Scale bars represent 100 μm; original magnification ×100. (c) Immunofluorescence staining of TGF‐β1 in DMSO‐treated and untreated wound tissue (n = 6). Scale bars represent 50 μm; original magnification ×200. (d) Migration of keratinocytes treated as indicated. MF, cocultured with mouse fibroblasts by a transwell assay; 1D11, a TGF‐β1 neutralizing antibody. Data are presented as the mean ± SEM with n = 5 for all groups, analysed by one‐way ANOVA with Kruskal–Wallis test for multiple comparison. Significance level: *P < 0.05, compared with the MF group, (e) western blot of migration‐related proteins in the keratinocytes treated as indicated

Article Snippet: According to the stimulants added to the medium in the bottom chamber, six experimental groups were established: PBS control, DMSO (5 mM), recombinant mouse TGF‐β1 (5 ng·ml −1 , R&D Systems, Minnesota, USA), mouse fibroblasts (MFs, 4 × 10 5 cells), MF + DMSO and MF + DMSO+1D11 (a TGF‐β1 (TGFBR1) neutralizing antibody, 10 g·ml −1 , R&D Systems, Minnesota, USA).

Techniques: Migration, Derivative Assay, Staining, Immunofluorescence, Transwell Assay, Western Blot

Journal: Biology of reproduction

Article Title: Transforming growth factor beta1 from endometriomas promotes fibrosis in surrounding ovarian tissues via Smad2/3 signaling.

doi: 10.1093/biolre/iox140

Figure Lengend Snippet: Figure 6. Analyzing the fibrotic effects of TGF-β1 on EdSCs in vitro. After EdSCs were exposed with TGF-β1, fibrosis-related genes including COL1A1, ACTA2, CTGF, and FN1 at 4, 12, 24, 48, and 72 h were detected by RT-qPCR (A), and fibrosis-related markers including COL1A1, ACTA2, CTGF, and MMP2 were analyzed by western blotting (B). The components’ expression of the TGF-β1/Smad signaling pathway such as SMAD2, p-SMAD2, SMAD3, and p-SMAD3 were demonstrated by western blotting analysis (C).

Article Snippet: Briefly, a series of 5-μm sections was incubated with the following antibodies: monoclonal rabbit antibodies against human TGFB1 (1:200), TGF-β1 receptor (TGFBR1; 1:500; sc-398; Santa Cruz Biotechnology), SMAD2 (1:200), SMAD3 (1:200), SMAD4 (1:200), SMAD2/3 (1:200), phospho-SMAD2 (p-SMAD2; 1:200), phospho-SMAD3 (p-SMAD3; 1:200), Collagen I (COL1A1, 1:200), connective tissue growth factor (CTGF, 1:1000), alpha-smooth muscle actin (ACTA2, 1:200), cluster of differentiation 10 (CD10, 1:200; ab126593; Abcam), and a monoclonal mouse antibody against human pan cytokeratin (KRT (pan); 1:200; ab7753; Abcam).

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

Journal: Biology of reproduction

Article Title: Transforming growth factor beta1 from endometriomas promotes fibrosis in surrounding ovarian tissues via Smad2/3 signaling.

doi: 10.1093/biolre/iox140

Figure Lengend Snippet: Figure 7. IHC detection of activated TGF-β1/Smad signaling in the cystic walls of endometriomas. (A) Representative photomicrograph of an excised endometri- oma stained with Masson’s trichrome after laparoscopic cystectomy. The red lines separated ovarian tissue and the cystic wall, while yellow lines separated the endometriotic loci and the cystic wall. Follicles were highlighted with arrows in (A) and identify ovarian tissue. The expression of TGFB1 (B) and TGFBR1 (C), and fibrosis-related factors including ACTA2 (D), COL1A1 (E), and CTGF (F), and components of the TGF-β1/Smad signaling pathway including p-SMAD2 (G), p-SMAD3 (H), SMAD2/3 (I) SMAD2 (J), SMAD3 (K), and SMAD4 (L) were demonstrated in the serial sections. (M) A comparison among the percentage of positive expression areas of different primary antibody in the cystic walls of endometriomas. Magnified photomicrographs with high-resolution view areas of the blue-square frames were shown in Supplemental Figure S4 correspondingly. (A–L: 100×).

Article Snippet: Briefly, a series of 5-μm sections was incubated with the following antibodies: monoclonal rabbit antibodies against human TGFB1 (1:200), TGF-β1 receptor (TGFBR1; 1:500; sc-398; Santa Cruz Biotechnology), SMAD2 (1:200), SMAD3 (1:200), SMAD4 (1:200), SMAD2/3 (1:200), phospho-SMAD2 (p-SMAD2; 1:200), phospho-SMAD3 (p-SMAD3; 1:200), Collagen I (COL1A1, 1:200), connective tissue growth factor (CTGF, 1:1000), alpha-smooth muscle actin (ACTA2, 1:200), cluster of differentiation 10 (CD10, 1:200; ab126593; Abcam), and a monoclonal mouse antibody against human pan cytokeratin (KRT (pan); 1:200; ab7753; Abcam).

Techniques: Staining, Expressing, Comparison