Journal: Oncoimmunology

Article Title: Systemic administration of β-glucan of 200 kDa modulates melanoma microenvironment and suppresses metastatic cancer.

doi: 10.1080/2162402X.2017.1387347

Figure Lengend Snippet: Figure 3. BG34-200 treatment augments systemic activation of T cells recognizing melanoma antigen. (A) Splenocytes or TDLN cells were stimulated with gp10025-33 peptide. IFN-g and IL-2 levels in the culture media were assessed using ELISA. B. Frequencies of CD3, CD4, CD8 T cells and their expression of CD62L and CD44 in spleen and TDLN were determined by FACS analysis. C. TDLN cells were subjected to intracellular cytokine staining for determining the frequency of gp10025-33 -specific T cells. (A) and (B) were graphed as means ± SD. Each data point represents one of three replicates of samples from individual TDLN or spleen. * p < 0.05, ** p < 0.01. Significance was determined using Student’s t test.

Article Snippet: Mouse IFN- and IL-2 ELISA kits were purchased from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Staining

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: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Targeting S1P1 receptor protects against murine immunological hepatic injury through myeloid-derived suppressor cells.

doi: 10.4049/jimmunol.1301193

Figure Lengend Snippet: FIGURE 2. Treatment with FTY720 increases the liver expression of chemokines that mediate CD11b+Gr1+ myeloid cell recruitment. (A–D) C57BL/6 mice were injected with PBS or FTY720 (1 mg/kg) daily starting at 6 h before the injection of Con A (15 mg/kg). (A) At 20 h after Con A injection, the percentage of peripheral blood CD11b+Gr1+ cells was determined by flow cytometry, and the absolute numbers of CD11b+Gr1+ cells were calculated accordingly (n = 5–8). (B) CD11b+Gr1+ cells in liver were determined by flow cytometry and are presented as a representative FACS plot (left panel) and the absolute numbers of CD11b+Gr1+ cells (n = 6–8) (right panel). (C) The concentration of CXCL1 and CXCL2 in serum (left panel) or the mRNA level of CXCL1 and CXCL2 in liver (right panel) CD11b+Gr1+ cells was determined by ELISA (n = 4) or quantitative PCR (n = 4). (D) CXCR2 expression in liver CD11b+Gr1+ cells was determined by intracellular staining, followed by flow cytometry analysis (n = 3). (E and F) CIH mice were administered 50 mg anti-CXCR2 mAb (242216) or IgG isotype control (54447; both from R&D Systems) via i.v. injection. (E) Mice were sacrificed at 20 h after Con A injection, and livers were analyzed using H&E staining. Scale bar, 50 mm. Arrowheads indicate the area of necrosis. (F) The percentage of necrotic area (left panel) was quantified using ImageJ software. For each experiment, three random sections/liver were taken from four livers/group. Serum ALT levels were assessed by ELISA (n = 4) (right panel). Data are representative of four (A, B) or two (C–F) independent experiments (mean 6 SD). *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: The concentrations of serum CXCL1 and CXCL2 and serum or culture supernatant IFN-g were quantified by sandwich ELISA (MKC00B, MM200, and MIF00; R&D Systems).

Techniques: Expressing, Injection, Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Staining, Control, Software

Journal: Arthritis & rheumatology (Hoboken, N.J.)

Article Title: Imbalance Between Bone Morphogenetic Protein 2 and Noggin Induces Abnormal Osteogenic Differentiation of Mesenchymal Stem Cells in Ankylosing Spondylitis.

doi: 10.1002/art.39433

Figure Lengend Snippet: Figure 4. Levels of bone morphogenetic protein 2 (BMP-2) and Noggin secreted by AS-MSCs compared with HD-MSCs during osteogenic dif- ferentiation. A and B, BMP-2 (A) and Noggin (B) protein levels in HD-MSCs and AS-MSCs were detected by enzyme-linked immunosorbent assay from day 0 to day 21 of induction. C, Top, Activation levels of the Smad1/5/8, p38 MAPK, JNK, and ERK-1/2 signaling pathways in HD- MSCs and AS-MSCs were determined by Western blotting. Bottom, Results of Western blotting were quantified as the mean 6 SD intensity ratio of phosphorylated to nonphosphorylated proteins. Values are the mean 6 SD of 12 samples per group. * 5 P , 0.05. See Figure 1 for other definitions.

Article Snippet: In addition, BMP-2 and Noggin were measured in the serum using a Quantikine ELISA kit for BMP-2 (R&D Systems) and an ELISA kit for human Noggin (Uscn Life Science).

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Protein-Protein interactions, Western Blot

Journal: Cardiology in the Young

Article Title: Identifying kidney injury via urinary biomarkers after the comprehensive stage II palliation and bidirectional Glenn procedure: a pilot study

doi: 10.1017/s1047951125100838

Figure Lengend Snippet: Figure 2. Changes in biomarker levels over time and ratios of actual biomarker values between comprehensive stage II (Comp II) and bidirectional Glenn (Glenn) palliation. Comparison of (A) urinary neutrophil gelatinase-associated lipocalin (NGAL), (B) urinary liver fatty acid-binding protein (L-FABP), (C) urinary kidney molecular injury - 1 (KIM-1), (D) Interleukin-18 (IL-18) and (E) Cystatin C between patients undergoing Comp II and Glenn palliation pre-operatively, followed by 1, 6 and 24 hours post-operatively. Data are presented as the natural logarithm (ln) transformed values (except data for interleukin-18). Linear mixed model was used for statistical analysis. A’ to E’: Ratios of the actual (non- transformed) values between the Comp II and the Glenn palliation are presented with their 95% confidence interval. A confidence interval not encompassing 1 for ratios indicates a statistically significant difference (denoted by *) between groups.

Article Snippet: Urinary concentrations of neutrophil gelatinase-associated lipocalin (R&D system, catalog number DLCN20), interleukin-18 (R&D system, catalog number DBP180), liver fatty acid-binding protein (Abcam, catalog number ab218261), kidney injury molecule-1(R&D system, catalog number DKM100), and cystatin C (R&D system, catalog DSCTCO) were determined using commercially available enzyme-linked immunosorbent assay kits and normalised to urinary creatinine (Cayman Chemical, catalog number 500701).

Techniques: Biomarker Discovery, Comparison, Binding Assay, Transformation Assay