Journal: Molecules and cells

Article Title: Transforming Growth Factor-β-Induced RBFOX3 Inhibition Promotes Epithelial-Mesenchymal Transition of Lung Cancer Cells.

doi: 10.14348/molcells.2016.0150

Figure Lengend Snippet: Fig. 3. Changes in the expression of EMT-related proteins following RBFOX3- depletion. (A) TGF-β1 treatment in- duced EMT in A549 cells. A549 cells were treated with TGF-β1 (5 ng/ml) for 48 h, and then cell lysates were sub- jected to immunoblot analysis for the indicated proteins. (B, C) Immunoblot analysis of EMT-related proteins. Con- struct of CRISPR-Cas9 for RBFOX3- KD were transfected into A549 cells. At 24 h after transfection, cells were treat- ed with TGF-β1 (5 ng/ml) for 48 h. The cell lysates were subjected to immunob- lot analysis for the indicated proteins. The values under the gels denote rela- tive intensities of the protein bands.

Article Snippet: Neutralizing anti-TGF-β antibody (1D11) and human TGF-β1 (R&D Systems, USA) were supplemented into the culture medium at concentrations of 25 μg/ml and 5 ng/ml, respectively.

Techniques: Expressing, Western Blot, CRISPR, Transfection

Journal: Molecules and cells

Article Title: Transforming Growth Factor-β-Induced RBFOX3 Inhibition Promotes Epithelial-Mesenchymal Transition of Lung Cancer Cells.

doi: 10.14348/molcells.2016.0150

Figure Lengend Snippet: Fig. 4. RBFOX3 depletion-induced morpho- logical changes in EMT phenotype. (A) RBFOX3-KD cells were treated with TGF- β1 (5 ng/ml) for 24 h. The fixed and perme- abilized cells were immunostained for the indicated proteins. Scale bars indicate 10 μm. (B) Quantification of NMHC II-A-stained cytoskeletal fibers. Each bar represents the number of NMHC II-A-stained cytoskeletal fibers in the cytoplasm (mean values ± SD, 10 cells). (C) Quantification of Vimentin- stained cytoskeletal fibers. Each bar repre- sents the intensity of Vimentin-stained cyto- skeletal fibers in the cytoplasm (mean val- ues ± SD, 10 cells). *P < 0.05 (two-sided t test) compared with control cells. (D) A model for the RBFOX3-mediated regulation of EMT processing. RBFOX3 expression is tran- scriptionally inhibited by TGF-β1. RBFOX3- inhibition stimulates morphological EMT phenotype.

Article Snippet: Neutralizing anti-TGF-β antibody (1D11) and human TGF-β1 (R&D Systems, USA) were supplemented into the culture medium at concentrations of 25 μg/ml and 5 ng/ml, respectively.

Techniques: Staining, Control, Expressing, Inhibition

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: BMC Developmental Biology

Article Title: Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells

doi: 10.1186/1471-213X-7-7

Figure Lengend Snippet: TGF-β is the acid-activated serum factor that induces branching tubulogenesis. J3B1A cells were grown in collagen gels in defined medium for 6 days to allow the formation of cystic structures. The cultures were then left untreated or were incubated with 1% acidified FCS (pH 3) for an additional 48 hours. (A) Under control conditions, J3B1A cells form spheroidal cysts enclosing a wide lumen. (B) Addition of acidified FCS induces the radial outgrowth of tube-like structures from the cyst wall. (C) Co-addition of 5 μM SB-431542, a selective inhibitor of the TGF-β type I receptor, abolishes the tubule-inducing activity of acidified FCS. (D) Pre-incubation of acid-treated FCS with a neutralizing antibody specific for TGF-β1 abrogates the tubulogenic effect of acidified FCS, whereas pre-incubation with a neutralizing antibody to TGF-β2 (E) or with a control antibody that does not react with TGF-βs (F) have no inhibitory effect. Antibodies in D-F were added at a final concentration of 5 μg/ml. Bars, 200 μm. (G) Branching tubulogenesis is suppressed by SB-431542 in a dose-dependent manner. The cultures were treated with the inhibitor two hours before addition of 1% acidified FCS. Data were expressed as mean number of outgrowths per colony ± s.e.m. from three separate experiments and statistical significance was determined using the Student's unpaired t -test. * p < 0.0025 versus values of acidified FCS alone. (H) Dose-response analysis of the effect of increasing concentrations of anti-TGF-β1 antibody. Acidified FCS (1%) was pre-incubated for 60 minutes with the indicated concentrations of the antibody before being added to the cultures. * p < 0.025 versus values of acidified FCS alone. ** p < 0.0005 versus values of acidified FCS alone.

Article Snippet: Chicken neutralizing antibody (IgY) to TGF-β1 (Cat. AB-101-NA), control chicken IgY that do not react with TGFβ1 (Cat. AB-101-C), and goat neutralizing antibody to TGF-β2 (Cat. AB-112-NA) were purchased from R&D Systems.

Techniques: Incubation, Control, Activity Assay, Concentration Assay

Journal: BMC Developmental Biology

Article Title: Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells

doi: 10.1186/1471-213X-7-7

Figure Lengend Snippet: Low concentrations of exogenous TGF-β1 induce morphogenesis of branching tubules. (A) J3B1A cells grown in a collagen gel in defined medium for a total of 10 days. (B) Parallel culture in which J3B1A cells were grown in a collagen gel for 6 days to allow cyst formation and were subsequently treated with 50 pg/ml TGF-β1 for an additional 4 days. TGF-β1 has induced the outgrowth of tube-like structures from the wall of existing cysts. (C) Treatment with 2 ng/ml TGF-β1 has resulted in the formation of numerous thin cell cords extending out into the surrounding collagen matrix. Notably, at this relatively high concentration, TGF-β1 also disrupts the organization of preformed cysts, resulting in lumen obliteration. (D) Higher magnification view of a multicellular structure formed in a culture treated with 20 pg/ml TGF-β1 for 4 days. The outgrowths enclose a patent lumen, which at least in some tubes is continuous with the cavity of the cyst. (E) Semi-thin section of a collagen gel culture of J3B1A cells treated with 50 pg/ml TGF-β1 for 4 days. Bars (A-E), 200 μm. (F) Quantitative analysis of TGF-β1-induced tubulogenesis. J3B1A cells were grown in a collagen gel for 6 days to allow cyst formation and were subsequently treated with different concentrations of TGF-β1. Tube formation was evaluated as described in Materials and Methods after 4 days of treatment. Data were expressed as mean number of outgrowths per colony ± s.e.m. from three separate experiments. p < 0.0005 for values of 20 pg/ml TGF-β1 at 2 days compared with control at 2 days, as well as for values of 100 pg/ml TGF-β1 at 2 days compared with 50 pg/ml TGF-β1 at 2 days; p < 0.0025 for values of 50 pg/ml TGF-β1 at 4 days compared with 2 days; p < 0.01 for values of 50 pg/ml TGF-β1 at 4 days compared with 20 pg/ml TGF-β1 at 4 days; p < 0.025 for values of 50 pg/ml TGF-β1 at 2 days compared with 20 pg/ml TGF-β1 at 2 days, as well as for values of 20 pg/ml TGF-β1 at 4 days compared with 2 days.

Article Snippet: Chicken neutralizing antibody (IgY) to TGF-β1 (Cat. AB-101-NA), control chicken IgY that do not react with TGFβ1 (Cat. AB-101-C), and goat neutralizing antibody to TGF-β2 (Cat. AB-112-NA) were purchased from R&D Systems.

Techniques: Concentration Assay, Control

Journal: BMC Developmental Biology

Article Title: Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells

doi: 10.1186/1471-213X-7-7

Figure Lengend Snippet: TGF-β1-induced tubulogenesis in fibrin gels. (A) J3B1A cells grown in a fibrin gel in defined medium for 11 days have formed spherical cysts. (B, C) J3B1A cells grown in a fibrin gel in defined medium for 4 days and subsequently treated with 20 pg/ml (B) or 100 pg/ml (C) TGF-β1 for an additional 7 days have formed branched tubules. (D) Cells grown as in (A-C) but incubated with 500 pg/ml TGF-β1 have generated a complex network of solid anastomosing cords. Bar, 200 um. (E) Quantitative analysis of TGF-β1-induced tubulogenesis in fibrin gels. J3B1A cells were grown in a fibrin gel for 6 days to allow cyst formation and were subsequently treated with different concentrations of TGF-β1. Tube formation was evaluated as described in Materials and Methods after 2, 4 and 7 days of treatment. Data were expressed as mean number of outgrowths per colony ± s.e.m. from three independent experiments. p < 0.0005 for values of 20 pg/ml TGF-β1 at 2 days compared with control at 2 days, as well as for values of 50 pg/ml and 100 pg/ml TGF-β1 at 7 days compared with 2 days; p < 0.005 for values of 20 pg/ml TGF-β1 at 7 days compared with 2 days; p < 0.01 for values of 50 pg/ml TGF-β1 at 4 days compared with 2 days; p < 0.025 for values of 100 pg/ml TGF-β1 at 4 days compared with 2 days; p < 0.05 for values of 20 pg/ml TGF-β1 at 4 days compared with 2 days, as well as for values of 50 pg/ml TGF-β1 at 2 days compared with 20 pg/ml TGF-β1 at 2 days.

Article Snippet: Chicken neutralizing antibody (IgY) to TGF-β1 (Cat. AB-101-NA), control chicken IgY that do not react with TGFβ1 (Cat. AB-101-C), and goat neutralizing antibody to TGF-β2 (Cat. AB-112-NA) were purchased from R&D Systems.

Techniques: Incubation, Generated, Control

Journal: BMC Developmental Biology

Article Title: Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells

doi: 10.1186/1471-213X-7-7

Figure Lengend Snippet: Suppression of TGF-β1-induced tubulogenesis by a p38 inhibitor. (A-C) J3B1A cells were grown in collagen gels for 6 days and subsequently treated with 50 pg/ml TGF-β1 alone (A), co-treated with TGF-β1 and U0126, an inhibitor of the ERK activators MEK1 and MEK2 (B), or co-treated with TGF-β1 and PD169316, a p38 inhibitor (C), for 4 days. The inhibitors were added two hours before treatment with TGF-β1. Whereas relatively high concentrations (20 μM) of U0126 only attenuate TGF-β1-induced tubulogenesis, PD169316 has a profound inhibitory effect at a concentration as low as 5 μM. Bars, 200 μm. (D) Quantitative analysis of inhibition of tubulogenesis. Data were expressed as mean number of outgrowths per colony ± s.e.m. from at least three separate experiments, and statistical significance was determined using the Student's unpaired t -test. p < 0.01 and p < 0.0005 for values of 5 μM and 10 μM U0126, respectively, compared with cultures incubated with TGF-β1 alone (Ctrl). p < 0.05 and p < 0.0005 for values of 5 μM and 10 μM SP 600125, respectively, compared with cultures incubated with TGF-β1 alone (Ctrl). p < 0.0005 for values of 2 μM PD169316 compared with cultures incubated with TGF-β1 alone (Ctrl).

Article Snippet: Chicken neutralizing antibody (IgY) to TGF-β1 (Cat. AB-101-NA), control chicken IgY that do not react with TGFβ1 (Cat. AB-101-C), and goat neutralizing antibody to TGF-β2 (Cat. AB-112-NA) were purchased from R&D Systems.

Techniques: Concentration Assay, Inhibition, Incubation

Journal: BMC Developmental Biology

Article Title: Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells

doi: 10.1186/1471-213X-7-7

Figure Lengend Snippet: TGF-β1 induces the production of MMP-9 by J3B1A cells. (A) Western blot analysis of conditioned media from J3B1A cells incubated with 50, 200 and 1000 pg/ml TGF-β1 for 72 hours. MMP-9 is induced by TGF-β1 in a dose-dependent manner. MMP-13 and MT1-MMP (MMP-14) are constitutively expressed by J3B1A cells and are not ostensibly modulated by TGF-β1. Conditioned medium from PMA-treated human U937 cells was used as a positive control for MMP-9, and conditioned medium from SVEC4-10 cells as a control for MMP-13 and MMP-14. Uniform loading of lanes was verified by silver staining. The blots shown are representative of at least two independent experiments. (B) Gelatin zymography of conditioned media from J3B1A cells incubated with 50, 200 and 1000 pg/ml TGF-β1 for 72 hours. TGF-β1 induces the secretion of MMP-9 in a dose-dependent manner, a clear increase in MMP-9 activity being already evident at 50 pg/ml TGF-β1. Conditioned medium from PMA-treated U937 cells, which are known to produce MMP-2 and MMP-9, was used as a positive control.

Article Snippet: Chicken neutralizing antibody (IgY) to TGF-β1 (Cat. AB-101-NA), control chicken IgY that do not react with TGFβ1 (Cat. AB-101-C), and goat neutralizing antibody to TGF-β2 (Cat. AB-112-NA) were purchased from R&D Systems.

Techniques: Western Blot, Incubation, Positive Control, Control, Silver Staining, Zymography, Activity Assay

Journal: BMC Developmental Biology

Article Title: Low concentrations of transforming growth factor-beta-1 induce tubulogenesis in cultured mammary epithelial cells

doi: 10.1186/1471-213X-7-7

Figure Lengend Snippet: Effect of MMP inhibitors on TGF-β1-induced tubulogenesis. (A-C) The broad spectrum metalloproteinase inhibitor BB94 abrogates TGF-β1-induced branching tubulogenesis. J3B1A cells were grown in a collagen gel for 6 days to allow cyst formation and were subsequently treated with 50 pg/ml TGF-β1 for an additional 4 days in the absence (A) or in the presence (B) of BB94 (1 μM; the inhibitor was added two hours before treatment with TGF-β1). Bars, 200 μm. (C) Quantitative analysis of the effect of BB94 on TGF-β1-induced tubulogenesis. J3B1A cells grown in collagen gels for 6 days were treated with TGF-β1 alone (50 pg/ml), co-treated with TGF-β1 and BB94 (30 nM to 3 μM), or co-treated with TGF-β1 and the inactive isomer BB1268 (3 μM) for 4 days. Formation of tubular outgrowths is suppressed in a dose-dependent manner by BB94, but is not significantly decreased by the inactive isomer BB1268. * p < 0.0125 and ** p < 0.0005 compared with cultures incubated with TGF-β1 alone. (D-F) TGF-β1-mediated branching tubulogenesis is suppressed by pre-treatment with 2 μg/ml recombinant TIMP-2 or 5 μM MMP-9 Inhibitor I, but not by pre-treatment with 100 μM CL-82198, a selective inhibitor of MMP-13. Bars, 200 μm. (G) Quantitative analysis of the effect of MMP-9 inhibitor I. J3B1A cells grown in collagen gels for 6 days were treated with TGF-β1 alone (50 pg/ml) or co-treated with TGF-β1 and MMP-9 Inhibitor I (1–5 μM) for 4 days (MMP-9 Inhibitor I was added two hours before treatment with TGF-β1). Formation of tubular outgrowths is abrogated by MMP-9 inhibitor I in a dose-dependent manner. * p < 0.0025 and ** p < 0.0005 compared with cultures incubated with TGF-β1 alone.

Article Snippet: Chicken neutralizing antibody (IgY) to TGF-β1 (Cat. AB-101-NA), control chicken IgY that do not react with TGFβ1 (Cat. AB-101-C), and goat neutralizing antibody to TGF-β2 (Cat. AB-112-NA) were purchased from R&D Systems.

Techniques: Incubation, Recombinant