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: Nucleic Acids Research

Article Title: High-efficiency homology-directed insertion into the genome using the engineered homing endonuclease ARCUS

doi: 10.1093/nar/gkaf961

Figure Lengend Snippet: Highly efficient and specific small insertions, deletions, and substitutions are enabled using ARCUS and AAV. T cells were electroporated with mRNA encoding a TGFBR2 -specific ARCUS nuclease and transduced with one of four AAV6 templates displaying 500 bp HAs flanking the cut site. Each AAV6 vector in this study contained mismatches in the RHA as denoted in ( A ). Representative histograms shown in ( B ) show loss of TGFBRII display on insert + cells, compared to cells treated with repair vector or nuclease alone. Data are representative of three independent experiments using T cells from two human donors.

Article Snippet: At 7 days post electroporation, single-cell suspensions were labeled with antibodies against human CD3 (BioLegend, San Diego, CA, Cat# 344818), TGFBRII (R&D Systems, Minneapolis, MN, Cat# AF-241-NA), or B2M (Becton-Dickinson, Franklin Lakes, NJ, Cat# 551337).

Techniques: Transduction, Plasmid Preparation

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