Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Separation of cell survival, growth, migration, and mesenchymal transdifferentiation effects of fibroblast secretome on tumor cells of head and neck squamous cell carcinoma

doi: 10.1177/1010428317705507

Figure Lengend Snippet: FIB and SCC-25 cells produce TGF-β1 and the effects of TGF-β1 on SCC-25 cell proliferation and migration. (a) The TGF-β1 concentrations in the supernatants were measured using ELISA after 3 days of treatment. SCC-25 cells treated with 5 ng/mL TGF-β1 contained 917.1 ± 38.12 pg/mL in the supernatant. In the supernatant of the albumin-medium-treated (control) SCC-25 cells, there was almost no detectable TGF-β1 (0.3 ± 0.12 pg/mL). Significant levels of TGF-β1 were measured in the supernatants of FIB CM (164.1 ± 6.85 pg/mL) and mixed-culture CM (106.0 ± 5.82 pg/mL)-treated SCC-25 cells, whereas the TGF-β1 levels in co-culture (2.3 ± 0.94 pg/mL) were low. In the supernatant of SCC-25-CM-treated cells 10.4 ± 2.53 pg/mL TGF-β1 was measured. (b) After treatment with 5 ng/mL TGF-β1, there were significantly (p < 0.01) less SCC-25 cells growing than in the albumin-medium treated (control). (c) 0–96 h treatment with 5 ng/mL TGF-β1 leads to no change in coverage of the scraped area, while in the albumin-medium treated (control; p < 0.001), it was decreased. (d) 48–96 h treatment with 5 ng/mL TGF-β1 cells were also distributed over the scraped area (CM: conditioned medium; FIB: human gingival fibroblasts; **p < 0.01; ***p < 0.001). Cells treated with 5 ng/mL TGF-β1 showed elongated, mesenchymal-like morphology at 72–96 h (bars: 100 μm).

Article Snippet: Then, the cells were washed twice with DPBS and treated for 72 h with 2 mL/well albumin-supplied or CM by exchanges every 24 h. After completion of treatments, the supernatants were taken from the SCC-25 cells and frozen at −20°C to be used for the human TGF-β1 PicoKine™ enzyme-linked immunosorbent assay (ELISA Kit; Boster Biological Technology, Pleasanton, CA, USA) later on.

Techniques: Migration, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Separation of cell survival, growth, migration, and mesenchymal transdifferentiation effects of fibroblast secretome on tumor cells of head and neck squamous cell carcinoma

doi: 10.1177/1010428317705507

Figure Lengend Snippet: Fold change EMT-related gene expressions in SCC-25 cells related to the albumin-medium-treated control.

Article Snippet: Then, the cells were washed twice with DPBS and treated for 72 h with 2 mL/well albumin-supplied or CM by exchanges every 24 h. After completion of treatments, the supernatants were taken from the SCC-25 cells and frozen at −20°C to be used for the human TGF-β1 PicoKine™ enzyme-linked immunosorbent assay (ELISA Kit; Boster Biological Technology, Pleasanton, CA, USA) later on.

Techniques:

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Separation of cell survival, growth, migration, and mesenchymal transdifferentiation effects of fibroblast secretome on tumor cells of head and neck squamous cell carcinoma

doi: 10.1177/1010428317705507

Figure Lengend Snippet: Fold change of vimentin and E-cadherin protein related to the control. The protein synthesis of vimentin, E-cadherin and β-actin in SCC-25 cells was determined using western blot analysis. β-actin was applied as a loading control. Fibroblasts (a, lane 7) served as positive control cells for vimentin, since this protein was contained in undetectable levels in parental SCC-25 cells. (a) Typical western blot of 57 kDa vimentin, 120 kDa E-cadherin, and 42 kDa β-actin in samples: (1) control SCC-25 treated with albumin medium; (2) SCC-25 treated with FIB CM; (3) SCC-25 treated with SCC-25 CM; (4) SCC-25 treated with mixed-culture CM; (5) SCC-25 co-cultured with fibroblasts; (6) SCC-25 treated with TGF-β1 (5 ng/mL); and (7) cultured fibroblasts, positive control for vimentin and negative control for E-cadherin (CM: conditioned medium). In FIB and mixed-culture CM-treated SCC-25 cells, a faint vimentin band appeared. (b–c) The band intensities were analyzed with densitometry. The different treatments were related to control cells treated with albumin-containing medium only. Densitometry graphs covered 10 comparable western blot experiments. (b) Two times upregulation of vimentin was shown by treatment with FIB CM. (c) E-cadherin showed decrease in co-culture and after TGF-β1 treatment.

Article Snippet: Then, the cells were washed twice with DPBS and treated for 72 h with 2 mL/well albumin-supplied or CM by exchanges every 24 h. After completion of treatments, the supernatants were taken from the SCC-25 cells and frozen at −20°C to be used for the human TGF-β1 PicoKine™ enzyme-linked immunosorbent assay (ELISA Kit; Boster Biological Technology, Pleasanton, CA, USA) later on.

Techniques: Western Blot, Positive Control, Cell Culture, Negative Control, Co-Culture Assay

Journal: Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine

Article Title: Separation of cell survival, growth, migration, and mesenchymal transdifferentiation effects of fibroblast secretome on tumor cells of head and neck squamous cell carcinoma

doi: 10.1177/1010428317705507

Figure Lengend Snippet: Comparison of the main changes observed in the secretome or co-culture treatments of tumor cells.

Article Snippet: Then, the cells were washed twice with DPBS and treated for 72 h with 2 mL/well albumin-supplied or CM by exchanges every 24 h. After completion of treatments, the supernatants were taken from the SCC-25 cells and frozen at −20°C to be used for the human TGF-β1 PicoKine™ enzyme-linked immunosorbent assay (ELISA Kit; Boster Biological Technology, Pleasanton, CA, USA) later on.

Techniques: Migration, Expressing

Journal: Stem Cell Research & Therapy

Article Title: Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages: an in vitro approach to lung inflammation

doi: 10.1186/s13287-016-0398-9

Figure Lengend Snippet: Time-course and dose-response of lipopolysaccharide ( LPS )-induced cytokine production. Alveolar macrophages were seeded at 2 × 10 6 /mL and, after adherence, treated for 1, 24, 48, and 72 h in the presence or absence of LPS (10 and 100 ng/mL). The release of tumor necrosis factor alpha ( TNF - α ), interleukin-6 ( IL - 6 ), and transforming growth factor beta ( TGF - β ) were evaluated in conditioned medium by ELISA. Results are the mean of a minimum of four experiments ± SEM. Statistical analysis was performed by Tukey’s multiple comparison test, with * p < 0.05 versus control cells

Article Snippet: TNF-α, IL-6, and TGF-β1 measurements were made using commercially available ELISA kits (Genorise, Glen Mills, PA, USA) according to the manufacturer’s specifications.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison, Control

Journal: Stem Cell Research & Therapy

Article Title: Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages: an in vitro approach to lung inflammation

doi: 10.1186/s13287-016-0398-9

Figure Lengend Snippet: Effects of CM and MVs on the production of pro- and anti-inflammatory cytokines. Alveolar macrophages were seeded at 2 × 10 6 /mL and, after adherence, treated for 1, 24, 48, and 72 h in the presence or absence of lipopolysaccharide ( LPS ) (10 and 100 ng/mL) or of conditioned medium ( cond. medium ) or microvesicles. The release of tumor necrosis factor alpha ( TNF - α ), interleukin-6 ( IL - 6 ), and transforming growth factor beta ( TGF - β ) were evaluated in conditioned medium by ELISA. Results are the mean of a minimum of three experiments ± SEM. Statistical analysis was performed by Tukey’s multiple comparison test, with § p < 0.05 vs LPS-treated cells

Article Snippet: TNF-α, IL-6, and TGF-β1 measurements were made using commercially available ELISA kits (Genorise, Glen Mills, PA, USA) according to the manufacturer’s specifications.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: Stem Cell Research & Therapy

Article Title: Evaluation of amniotic mesenchymal cell derivatives on cytokine production in equine alveolar macrophages: an in vitro approach to lung inflammation

doi: 10.1186/s13287-016-0398-9

Figure Lengend Snippet: Effect of transforming growth factor beta ( TGF - β ) and neutralizing anti-TGF-β antibody on lipopolysaccharide ( LPS )-induced tumor necrosis factor alpha ( TNF - α ) production. a Alveolar macrophages were seeded at 2 × 10 6 /mL and, after adherence, treated in the presence or absence of amniotic mesenchymal cell CM and MVs. Basal release of TGF-β was assessed by ELISA after 24 and 48 h of incubation. Each dot represents the value of a single animal. b After adherence, cells were treated in the presence or absence of equine TGF-β (300 pg/mL) and LPS (100 ng/mL) for 24 h. Cell viability was assessed by MTT test and TNF-α by ELISA. c After adherence, cells were treated in the presence or absence of anti-TGF-β antibody (0.2 μg/mL) or control mouse IgG (0.2 μg/mL) or CM and MVs, or LPS (100 ng/mL) for 24 h. TNF-α release was assessed by ELISA. Results are the mean of a minimum of three experiments ± SEM. Statistical analysis was performed by Tukey’s multiple comparison test, with ** p < 0.01 versus relative control cells and §§ p < 0.01 versus LPS-treated cells. OD optical density

Article Snippet: TNF-α, IL-6, and TGF-β1 measurements were made using commercially available ELISA kits (Genorise, Glen Mills, PA, USA) according to the manufacturer’s specifications.

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Control, Comparison

Journal: International heart journal

Article Title: Ryanodine Receptor Type 2 Plays a Role in the Development of Cardiac Fibrosis under Mechanical Stretch Through TGFβ-1.

doi: 10.1536/ihj.16-572

Figure Lengend Snippet: Figure 2. Reduction of TGFβ1 expression by downregulation of RyR2in cardiomyocytes under mechani- cal stretch. A: Measurement of RyR2 mRNA level in cultured cardiomyocytes by Real-Time PCR. B: Expres- sion of RyR2 protein level in cardiomyocytes with Western Blotting. C: TGFβ1 in supernates of cultured cardiomyocytes was detected with ELISA analysis. D: Expression of TGFβ1 protein level in cardiomyocytes with Western Blotting. E: Measurement of tgfb1 mRNA level in cultured cardiomyocytes using Real-Time PCR. Values are expressed as mean ± SEM obtained from 3 independent experiments. shRyR2 indicates RyR2 knockdown lentiviral particle-infected cardiomyocytes; MS, cardiomyocytes under mechanical stretch; MS + ryr2 KD, RyR2 knockdown cardiomyocytes under mechanical stretch. *P < 0.05 versus control.

Article Snippet: ELISA assay: Supernates of cultured cardiomyocytes were collected and examined by Rat Transforming Growth Factor β1 ELISA Kit (CUSABIO BIOTECH, China) following the manufacturer’s instructions.

Techniques: Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Infection, Control

Journal: PLoS ONE

Article Title: S100A9 Interaction with TLR4 Promotes Tumor Growth

doi: 10.1371/journal.pone.0034207

Figure Lengend Snippet: A. Quantitative real time RT-PCR analysis (see ) of TGFβ RNA expression from CD11b + cells (>90% pure by FACS analysis) from the spleen of C57BL/6, S100A9 −/− and TLR4 −/− animals in the absence of, or 14 days after inoculation, with 50,000 EL4 lymphoma cells subcutaneously. The mean expression from 4 separate experiments is shown where the expression in the C57BL/6 controls have been set to 1. B. ELISA measurements of TGFβ serum levels of C57BL/6 (filled circles), RAGE −/− (filled triangles), S100A9 −/− (filled squares) and TLR4 −/− (filled diamonds) in the absence of, or 14 days after inoculation (open symbols), with 50,000 EL4 lymphoma cells subcutaneously. Statistical analysis using two-tailed t test *** p = 0.0008; * p = 0.039 and 0.0028, respectively. There was no statistical significant difference in TGFβ serum levels between EL4 inoculated C57BL/6 or RAGE −/− mice.

Article Snippet: The TGFβ1 Quantikine ELISA kit (Abnova Taipei, Taiwan) was utilized to determine serum TGFβ levels, as indicated.

Techniques: Quantitative RT-PCR, RNA Expression, Expressing, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: S100A9 Interaction with TLR4 Promotes Tumor Growth

doi: 10.1371/journal.pone.0034207

Figure Lengend Snippet: A. Binding of S100A9 to immobilized TLR4/MD2 complex is blocked by ABR-215050. Sensorgrams obtained after injection (2 min at 30 µL/min) of 50 nM S100A9 ± ABR-215050 over amine coupled TLR4/MD2 (density ∼2.3 kRU). Sensorgrams from top to bottom: S100A9 without competitor and with 3.91, 31.25 and 1,000 µM ABR-215050. Arrows indicate injection of sample (1); sample buffer - i.e. HBS-P containing 1 mM Ca 2+ and 10 uM Zn 2+ (2); and regeneration of surface with 3 M EDTA (3). B. Anti-tumor effect of ABR-215050 in EL4 tumors inoculated (s.c.) into wild type mice. The ABR-215050 was administrated in the drinking water at 30 mg/kg/day seven days/week from day 0 throughout the experiment. Each data point represents mean ± SEM (n = 10; p<0.01, Mann Whitney U). Control animals received only normal drinking water. The water intake of the animals was not affected by the presence of ABR-215050 in the drinking water. C. ELISA measurements of TGFβ serum levels day 20 in C57BL/6 animals inoculated with EL4 tumors and animals treated with 30 mg/kg/day of ABR-215050, as indicated (p = 0.0037, Student t test).

Article Snippet: The TGFβ1 Quantikine ELISA kit (Abnova Taipei, Taiwan) was utilized to determine serum TGFβ levels, as indicated.

Techniques: Binding Assay, Injection, MANN-WHITNEY, Control, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Smad2/3-Regulated Expression of DLX2 Is Associated with Radiation-Induced Epithelial-Mesenchymal Transition and Radioresistance of A549 and MDA-MB-231 Human Cancer Cell Lines

doi: 10.1371/journal.pone.0147343

Figure Lengend Snippet: (A) The levels of immunoreactive TGF-β1 were quantified from the cell culture medium by ELISA, as described in the Materials and methods (***P < 0.001, **P < 0.01, versus Ct). Ct, control cell; IR, irradiated cells. (B) Cells were transfected with 100 μM si-Smad2/3 or si-Ct, incubated at 37°C for 24 h. Then the cells were exposed to IR at 8Gy (A549) or 4Gy (MDA-MB-231) and incubated at 37°C for 24 h. Total RNA was isolated from the cells and subjected to real time PCR analysis. The graph represents the average of three independent experiments ±SE (***P < 0.001). (C) Cells were transfected with 100 μM si-Smad2/3 or si-Ct and incubated at 37°C for 24 h. Then the cells were exposed to IR at 8Gy (A549) or 4Gy (MDA-MB-231) and incubated at 37°C for 24 h. Subsequently, the cell lysates were subjected to western blot analysis. Three independent experiments obtained similar results. (D) Protein levels were quantified by densitometry. Data are represented as relative values to those of si-Ct after normalization with β-actin (***P < 0.001, **P < 0.01, *P < 0.05 versus si-Ct).

Article Snippet: A549 and MDA-MB-231 (5×10 5 cells/well) cells were exposed to IR at 8Gy, 4Gy and incubated at 37°C for 24 h. The level of TGF-β1 protein in culture supernatants was measured using a TGF-β1 ELISA kit (BD Biosciences, San Diego, USA) according to the manufacturer's instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Irradiation, Transfection, Incubation, Isolation, Real-time Polymerase Chain Reaction, Western Blot