Journal: Cells

Article Title: Overexpression and Activation of αvβ3 Integrin Differentially Affects TGFβ2 Signaling in Human Trabecular Meshwork Cells

doi: 10.3390/cells10081923

Figure Lengend Snippet: Differentially expressed genes in WTβ3 and/or CAβ3 cells that are associated with glaucoma. Except where noted by *, Log 2 FC values were reported in DGE analysis using EdgeR. * Genes that were filtered out due to low abundance did not originally have a FC determined (ND). Log 2 FC was calculated from the raw count data. The values in parentheses were originally calculated using EdgeR.

Article Snippet: ELISA analysis was performed using an R&D Systems Human TGF-beta 2 Quantikine ELISA Kit (R & D Systems, Minneapolis, MN), and the procedure was performed according to the manufacturer’s instructions.

Techniques: Migration, Activity Assay, Binding Assay, Ubiquitin Proteomics

Journal: Cells

Article Title: Overexpression and Activation of αvβ3 Integrin Differentially Affects TGFβ2 Signaling in Human Trabecular Meshwork Cells

doi: 10.3390/cells10081923

Figure Lengend Snippet: Differentially expressed genes in WTβ3 and/or CAβ3 cells that are associated with IOP regulation. Log 2 FC values were reported in DGE analysis using EdgeR.

Article Snippet: ELISA analysis was performed using an R&D Systems Human TGF-beta 2 Quantikine ELISA Kit (R & D Systems, Minneapolis, MN), and the procedure was performed according to the manufacturer’s instructions.

Techniques:

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 2. TGFβ induces TGFβ2 expression in GBM and non-GBM cell lines. A, qRT-PCR of TGFB 1 , TGFB 2 , and TGFB 3 in LN229 cells treated with TGFβ1 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, qRT-PCR of TGFB 2 in LN229 cells treated with TGFβ1, TGFβ2, and TGFβ3 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in culture supernatant from LN229 cells treated with TGFβ for 72 hours. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGF b 2 in LN229 cells treated with TGFβ1 and/or the TβRI inhibitor (TβRI inh.) LY-2109761 for 3 hours. GAPDH mRNA levels were used as an internal normalization control. *, P < 0.05, using the Student t test; data, mean ± SD. E, qRT-PCR of TGFB 2 in GBM and non-GBM cell lines treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 3. CREB1 regulates the autocrine induction of TGFβ2 by TGFβ. A, nucleotide sequence of the proximal region of the TGFB 2 promoter. The SBEs and CREB1 site (CRE) are indicated relative to the transcription start site. ClustalW sequence alignment for 3 animal species [ Homo sapiens ( H.s .), Pan troglodytes ( P.t. ), and Mus musculus ( M.m .)] shows the conservation of the binding sites. B, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an shRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, qRT-PCR of TGFB 2 and CREB1 in LN229 cells expressing an siRNA targeting CREB1 treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. D, immunoblot analysis and qRT-PCR of TGFβ2 in LN229 cells expressing ICER treated with TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. The molecular weights are shown.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Sequencing, Binding Assay, Quantitative RT-PCR, Expressing, shRNA, Control, Western Blot

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 5. PI3K and RSK regulate the TGFβ- mediated induction of TGFβ2 through CREB1. A, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with TGFβ for 3 hours and the PI3K inhibitor (inh) LY-294002 for 24 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. B, immunoblot analysis and qRT-PCR of TGFB2 in LN229 cells treated with increasing amounts of the RSK inhibitor BI-D1870 for 24 hours and TGFβ for 3 hours. GAPDH mRNA levels were used as an internal normalization control. ***, P < 0.005, using the Student t test; data, mean ± SD. C, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with TGFβ for 48 hours and the PI3K inhibitor for 72 hours. *, P < 0.05, using the Student t test; data, mean ± SD. D, secreted TGFβ2 protein levels determined by ELISA in LN229 cells treated with the RSK inhibitor BI-D1870 for 72 hours and TGFβ for 48 hours. *, P < 0.05, using the Student t test; data, mean ± SD.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Western Blot, Quantitative RT-PCR, Control, Enzyme-linked Immunosorbent Assay

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 6. TGFβ2 correlates with CREB1 expression in GBM patient tumors. A and B, graphs showing the correlation between CREB1 and TGFB 1 (B) or TGFB 2 (A) mRNA levels in patient GBM tumor samples. Data obtained from the REMBRANDT database. A Spearman test was used, and the correlation coeffi cient (ρ) and the two-tailed P value are shown. C, graph showing the correlation between p-CREB1 and TGFβ2 protein levels in tissue microarrays (TMA) from patient GBM samples. Not all spots were evaluable in all stainings. A Spearman test was used, and the correlation coeffi cient (ρ) and the two- tailed signifi cance are shown. Representative images from the TMAs are shown; scale bar, 50 μm. D, Kaplan–Meier curves showing the OS of patients with TGFB2 mRNA levels upregulated ≥3-fold and CREB1 mRNA levels upregulated ≥2-fold. Statistical signifi cance was assessed by the log-rank test. Data obtained from the REMBRANDT database.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Two Tailed Test

Journal: Cancer discovery

Article Title: Active CREB1 promotes a malignant TGFβ2 autocrine loop in glioblastoma.

doi: 10.1158/2159-8290.CD-14-0275

Figure Lengend Snippet: Figure 7. CREB1 regulates TGFβ2 expression in PDX models. A, scheme showing the experimental procedure. B, IHC of p-CREB1 and TGFβ2 from mouse tumors 60 days after inoculation with neurospheres expressing shRNAs targeting CREB1 and control shRNAs; scale bar, 50 μm (C). Kaplan–Meier survival curves from mice in B. D, the TGFβ2 malignant autocrine loop. In GBM, TGFβ collaborates with the PI3K and RSK pathways through a CREB1– SMAD3 transcriptional complex to induce TGFβ2 expression. This leads to the generation of an autocrine loop and accumulation of TGFβ2 in the tumor, hyperactivation of TGFβ, and tumor progression.

Article Snippet: For the quantitative determination of TGFβ2 protein levels secreted to the media, we used the Human TGFβ2 Quantikine ELISA Kit (R&D Systems) following the manufacturer’s specifi cations.

Techniques: Expressing, Paraffin-embedded Immunohistochemistry, Control

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: 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