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: Oncotarget

Article Title: Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

doi: 10.18632/oncotarget.23771

Figure Lengend Snippet: Figure 1: Comparison on the down-regulation of Moutan Cortex from different regions on protein expression levels of ICAM-1,TGF-β1 and FN. HBZY-1 mesangial cells were treated with 200 μg/mL AGEs in the presence or absence of Moutan Cortex (MC) extract of 200 μg/mL. Aminoguanidine of 10 μM was used as the positive control while BSA (200 μg/mL) as blank control. (A) Western blotting was performed to compare the protein expression levels. (B–D) The grayscale scan results of ICAM-1, TGF-β1 and FN. a, Control; b, 200 μg/mL AGEs; c, Positive control aminoguanidine group; “d-m” represent MC from Anhui, Guizhou, Zhejiang, Henan, Hunan, Hebei, Sichuan, Chongqing, Shandong, Gansu. Data are expressed as means ± SD, n = 3. ###P < 0.001 vs. BSA group; *P < 0.05, **P < 0.01 and ***P < 0.001 vs. AGEs group; $P < 0.05, $$P < 0.01 and $$$P < 0.001 vs. MC from Anhui group.

Article Snippet: Rabbit anti-mouse FN, TGF-β1 and ICAM-1 monoclonal antibodies were offered by Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Comparison, Expressing, Positive Control, Control, Western Blot

Journal: Oncotarget

Article Title: Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

doi: 10.18632/oncotarget.23771

Figure Lengend Snippet: Figure 2: Effect of Moutan Cortex from different regions on ICAM-1 and TGF-β1 protein expression levels in kidney of DN rats. After being treated with STZ and/or MC extract of 5g/kg or , immunohistochemistry was conducted to evaluate the expression levels of ICAM-1 (A) and TGF-β1 (B) of renal tissues. “a” represents normal control; “b” represents model group (DN rats); “c” represents Positive control 0.1 g/kg AG; “d-m” represent Gansu, Chongqing, Shangdong, Sichuan, Zhejiang, Anhui, Hunan, Guizhou, Hebei, Henan.

Article Snippet: Rabbit anti-mouse FN, TGF-β1 and ICAM-1 monoclonal antibodies were offered by Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Expressing, Immunohistochemistry, Control, Positive Control

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: MicroRNA-150 relieves vascular remodeling and fibrosis in hypoxia-induced pulmonary hypertension.

doi: 10.1016/j.biopha.2018.11.058

Figure Lengend Snippet: Fig. 4. Effect of miR-150 on pulmonary fibrosis of pulmonary hypertension rats. (A) Pulmonary fibrosis was detected by Masson’s staining (200× magnification). Scale bars, 100 μm. (B) The area of pulmonary fibrosis was calculated and shown. The mRNA expressions of TGF-β1 (C) and collagen I (D) in lung tissues were evaluated by qPCR. (E) The protein levels of TGF-β1 and collagen I in lung tissues were measured by western blot assay. (F)&(G) Relative grey values of the protein bands were shown. (H) The expressions of TGF-β1 and collagen I in lung tissues were detected by immunohistochemical staining (400× magnification). Scale bars, 50 μm. Data were presented as mean ± SD. **P < 0.01, ***P < 0.001 versus the indicated group.

Article Snippet: The membranes were blocked in 5% nonfat milk for 1 h at room temperature, then incubated with primary antibodies against TGF-β1 (1:400, BOSTER, China), Collagen I (1:300, BOSTER, China), p-AKTser473 (1:500, KeyGen, China), AKT (1:500, KeyGen, China), p-mTORser2481 (1:500, Sangon Biotech, China), mTOR (1:1000, Cell signaling Technology, USA), and β-actin (1:500, Bioss, China) at 4°C overnight, followed by incubation with HRP-labeled Goat AntiRabbit or Goat Anti-Mouse IgG (1:5000, Beyotime, China) at 37°C for 45min.

Techniques: Staining, Western Blot, Immunohistochemical staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: MicroRNA-150 relieves vascular remodeling and fibrosis in hypoxia-induced pulmonary hypertension.

doi: 10.1016/j.biopha.2018.11.058

Figure Lengend Snippet: Fig. 6. Effect of miR-150 on the expressions of fibrosis-related molecules. The mRNA expressions of TGF-β1 (A) and collagen I (B) in PASMCs were measured by qPCR. (C) The protein levels of TGF-β1 and collagen I in PASMCs were detected by western blot assay. (D)&(E) Relative grey values of the protein bands were shown. Data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 versus the indicated group.

Article Snippet: The membranes were blocked in 5% nonfat milk for 1 h at room temperature, then incubated with primary antibodies against TGF-β1 (1:400, BOSTER, China), Collagen I (1:300, BOSTER, China), p-AKTser473 (1:500, KeyGen, China), AKT (1:500, KeyGen, China), p-mTORser2481 (1:500, Sangon Biotech, China), mTOR (1:1000, Cell signaling Technology, USA), and β-actin (1:500, Bioss, China) at 4°C overnight, followed by incubation with HRP-labeled Goat AntiRabbit or Goat Anti-Mouse IgG (1:5000, Beyotime, China) at 37°C for 45min.

Techniques: Western Blot

Journal: Fibrogenesis & Tissue Repair

Article Title: L 59 TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

doi: 10.1186/s13069-015-0034-9

Figure Lengend Snippet: Establishment of a sandwich ELISA for detecting L 59 LAP-DPs. a Schematic diagram of the ELISA for L 59 LAP-DPs. In the ELISA, L 59 LAP-DPs are first captured by coated L59 antibodies (Ab) and then sandwiched by biotin-conjugated anti-LAP antibodies (αLAP-Ab), which form a complex with strep-AP. For detection, an enzyme substrate is added and absorbance at 405 nm was measured. b , c Incubation time- and PLK concentration-dependent increases in the absorbance of the samples containing LAP incubated with PLK. The LAP (final 25 nM) was digested with 12.5 nM (open triangle), 25 nM (open reverse triangle), and 50 nM (open square) PLK, or without PLK (open circle) for 0–90 min, and then the samples were diluted by 1/50 and subjected to the L 59 LAP-DP ELISA ( b ). Also, 25 nM rhLAP was digested with 50 nM PLK (open square) or PLN (cross mark), or PLK in the presence of 5 μM camostat mesilate (open diamond) ( c ). All data were presented as mean ± SD from two different experiments ​*p-value <0.05, **p-value <0.01, ***p-value<0.001 obtained comparing to corresponding control values

Article Snippet: rhLAP β1 and anti-human LAP β1 mouse monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Sandwich ELISA, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay, Control

Journal: Fibrogenesis & Tissue Repair

Article Title: L 59 TGF-β LAP degradation products serve as a promising blood biomarker for liver fibrogenesis in mice

doi: 10.1186/s13069-015-0034-9

Figure Lengend Snippet: Correlation among L 59 LAP-DPs and active TGF-β in the culture medium, and intracellular signal transduction. a , c The ×9CAGA-Luc-transformed CCL64 cells were cultured in PLK-added CM derived from HEK293T cells overexpressing hLTGF-β1. After 6 h, the levels of active TGF-β1 and L 59 LAP-DPs were determined by respective ELISAs ( a ), and the extent of TGF-β signaling was measured by luciferase activity in CCL64 cells ( c ). b , d The scatterplots between the levels of L 59 LAP-DPs and active TGF-β shown in a ( b ) and between increases in L 59 LAP-DP levels and increases in luminescence from the values obtained compared to basal levels in the absence of PLK shown in c ( d ). A significant positive correlation was seen ( b ) * p-value <0.05, ***p-value <0.001 obtained comparing to 0

Article Snippet: rhLAP β1 and anti-human LAP β1 mouse monoclonal antibodies were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Transduction, Transformation Assay, Cell Culture, Derivative Assay, Luciferase, Activity Assay

Journal: bioRxiv

Article Title: Microglia-derived TGF-β1 ligand maintains microglia homeostasis via autocrine mechanism and is critical for normal cognitive function in adult mouse brain

doi: 10.1101/2023.07.05.547814

Figure Lengend Snippet: (A) Timeline for using Cx3cr1 CreER driver to target myeloid cell inducible Tgfb1 loss (samples were harvested at 3 weeks post TAM). (B) Gating strategy for identifying the myeloid cells in single-cell suspension. Cells were first gated according to FSC-SSC, then restricted to singles cells and live cells. Myeloid cells were identified as CD45+ CD11b+. (C, D) FACS analyses of TGF-β expression by myeloid cells in the spleen (top histograms) and in the CNS (lower histograms). The fluorescence minus one (FMO) is represented in black histograms and TGF-β immunostaining is shown in red. TGF-β staining above the background (FMO) is shown in solid red and represents the percentage shown for each analysis. (E, F) Compilation of TGF-β expression by flow cytometry on myeloid cells from the spleen and CNS of WT and KO mice. (G) ELISA quantification (pg/µg total protein) from serum and tissue from the spleen and brain (n = 3 mice per group) showing no difference in TGF-β protein levels in serum or spleen of Cx3cr1CreER- tgfb1 iKO or control mice and that the brain TGF-β ligand levels are below the detection limit of the kit. Mean±SE, Student’s t-test (each data point represents a single animal).

Article Snippet: These antibodies included: CD45 (clone 30-F11), CD11b (clone M1/70) and TGF-β1 (clone TW7-16B4) (all from Biolegend).

Techniques: Suspension, Expressing, Fluorescence, Immunostaining, Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control

Journal: The Journal of Experimental Medicine

Article Title: Phenotype, Localization, and Mechanism of Suppression of CD4 + CD25 + Human Thymocytes

doi: 10.1084/jem.20020110

Figure Lengend Snippet: Cell contact–dependent suppressive activity of CD4 + CD25 + human thymocytes is abrogated by the combined action of anti–CTLA-4 and anti-TGFβ1 mAbs. (A) CD4 + CD25 − thymocytes were stimulated with irradiated T cell–depleted allogeneic PBMNCs in the lower chamber of a transwell plate in the absence or presence of CD4 + CD25 + thymocytes, placed in the same or in the top chamber. On day 5, cells present in the bottom chamber were harvested and their proliferation assessed by measuring 3 [H]TdR uptake. (B) CD4 + CD25 − thymocytes were stimulated with allogeneic irradiated T cell–depleted PBMNCs in the presence of different numbers of autologous purified CD4 + CD25 + thymocytes without (black columns) or with isotype control (IgG1+IgG2a) (gray columns), anti-CTLA-4 (hatched columns), or anti–TGF-β1 mAb (dotted columns), or mixtures of them (white columns). Cell proliferation was measured by assessment of 3 [H]TdR uptake. Mean values (±SD) obtained in four separate experiments are reported.

Article Snippet: The PE-conjugated anti-TNFR2 (22235.311, mouse IgG2a), the purified anti–TGF-β1 (9016.2, mouse IgG1) neutralizing mAb, TGF-βR2 (goat IgG) neutralizing Ab, and biotin-conjugated anti–TGF-β1 (chicken IgY) and anti-LAP (TGF-β1) (goat IgG) Abs were purchased from R&D Systems.

Techniques: Activity Assay, Irradiation, Purification, Control

Journal: The Journal of Experimental Medicine

Article Title: Phenotype, Localization, and Mechanism of Suppression of CD4 + CD25 + Human Thymocytes

doi: 10.1084/jem.20020110

Figure Lengend Snippet: CTLA-4 and TGF-β1–mediated suppression is due to the inhibition of IL-2R α-chain expression and is overcome by IL-15. (A) CFSE-labeled CD4 + CD25 − thymocytes were stimulated with irradiated allogeneic PB non-T cells in absence or presence of autologous CD4 + CD25 + thymocytes with anti–CTLA-4, anti–TGF-β1, anti–CTLA-4 plus anti–TGF-β1 or isotype-matched (IgG1 plus IgG2a) mAbs, and assessed by flow cytometry for proliferation and surface CD25 expression. One representative of three separate experiments is shown. (B) CD25 (left) and CD69 (right) expression by CD4 + CD25 − thymocytes cultured under the experimental conditions mentioned in (A). CD4 + CD25 − thymocytes without mAb added (black columns), or containing isotype control (gray columns), anti–TGF-β1 (dotted columns), anti–CTLA-4 (hatched columns), or anti–CTLA-4 plus anti–TGF-β1 (white columns) mAbs. Columns represent mean values (±SD) found in three separate experiments. (C) Effect of the addition of exogenous IL-2 or IL-15 on the proliferative response of CD4 + CD25 − thymocytes stimulated with irradiated allogeneic non-T cells in absence or presence of CD4 + CD25 + autologous thymocytes. Columns represent mean values (±SD) obtained in three separate experiments.

Article Snippet: The PE-conjugated anti-TNFR2 (22235.311, mouse IgG2a), the purified anti–TGF-β1 (9016.2, mouse IgG1) neutralizing mAb, TGF-βR2 (goat IgG) neutralizing Ab, and biotin-conjugated anti–TGF-β1 (chicken IgY) and anti-LAP (TGF-β1) (goat IgG) Abs were purchased from R&D Systems.

Techniques: Inhibition, Expressing, Labeling, Irradiation, Flow Cytometry, Cell Culture, Control