Journal: Experimental and Therapeutic Medicine

Article Title: Expression and significance of NELIN and SM22α in varicose vein tissue

doi: 10.3892/etm.2015.2170

Figure Lengend Snippet: Primer sequences used for reverse transcription polymerase chain reaction analysis of NELIN.

Article Snippet: TransZol Up and two-step reverse transcription polymerase chain reaction (RT-PCR) kits were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). β-actin upstream and downstream primers were synthesized by Takara Bio, Inc. (Shiga, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: Experimental and Therapeutic Medicine

Article Title: Expression and significance of NELIN and SM22α in varicose vein tissue

doi: 10.3892/etm.2015.2170

Figure Lengend Snippet: Primer sequences used for reverse transcription polymerase chain reaction analysis of SM22α.

Article Snippet: TransZol Up and two-step reverse transcription polymerase chain reaction (RT-PCR) kits were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). β-actin upstream and downstream primers were synthesized by Takara Bio, Inc. (Shiga, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: Experimental and Therapeutic Medicine

Article Title: Expression and significance of NELIN and SM22α in varicose vein tissue

doi: 10.3892/etm.2015.2170

Figure Lengend Snippet: Reverse transcription polymerase chain reaction analysis revealed weak mRNA expression of NELIN in the vascular smooth muscle cells (VSMCs) of the experimental group, but strong expression in the control group. (A) Expected length of the PCR products was 611 bp (NELIN) and 312 bp (β-actin). Lanes: N, control group; X, DNA marker DL 2000; and P, experimental group. (B) Downregulation of NELIN mRNA expression in the VSMCs from the varicose vein tissue. Data are presented as the mean ± standard deviation.

Article Snippet: TransZol Up and two-step reverse transcription polymerase chain reaction (RT-PCR) kits were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). β-actin upstream and downstream primers were synthesized by Takara Bio, Inc. (Shiga, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Standard Deviation

Journal: Experimental and Therapeutic Medicine

Article Title: Expression and significance of NELIN and SM22α in varicose vein tissue

doi: 10.3892/etm.2015.2170

Figure Lengend Snippet: Reverse transcription polymerase chain reaction analysis revealed weak mRNA expression of SM22α in the vascular smooth muscle cells (VSMCs) of the experimental group, but strong expression in the control group. (A) Expected length of the PCR products was 232 bp (SM22α) and 472 bp (GADPH). Lanes: N, control group; X, DNA marker DL 2000; and P, experimental group. (B) Downregulation of SM22α mRNA expression in the VSMCs from the varicose vein tissue. Data are presented as the mean ± standard deviation.

Article Snippet: TransZol Up and two-step reverse transcription polymerase chain reaction (RT-PCR) kits were purchased from Beijing TransGen Biotech Co., Ltd. (Beijing, China). β-actin upstream and downstream primers were synthesized by Takara Bio, Inc. (Shiga, Japan).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Marker, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Vascular endothelial growth factor (VEGF)- and epoetin alpha (EPOα)-mediated STAT-5 phosphorylation. Human umbilical vein endothelial cells (HUVECs) were treated with 25 ng/mL VEGF (10 min) or 1 IU/mL EPOα (5–10 and 30 min) under three different cell culture conditions. White bar: replacement of cell culture medium for 24 h with M199 without fetal bovine serum (FBS) and growth factors supplemented with 0.5% bovine serum albumin (BSA) and 0.2 mM orthovanadate. Gray bars: replacement of cell culture medium for 24 h with M199 without FBS and growth factors. Black bars: replacement of cell culture medium for 12 h with M199 medium without FBS and growth factors supplemented with 0.2 mM orthovanadate 30 min before the experiments. In each experimental condition, cell responsiveness was evaluated by assessing whether VEGF or EPOα stimulated STAT-5 phosphorylation. The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments. *** p < 0.001; ** p < 0.01 vs. basal value.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Concentration-dependence of EPOα-, darbepoetin alpha (DarbEPO)- and continuous erythropoietin receptors activator (CERA)-mediated STAT-5 phosphorylation. HUVECs were treated with different epoetin concentrations (0.5–10 IU/mL) for 5 min. STAT-5 phosphorylation levels were quantified using an enzyme-linked immunosorbent assay (ELISA) kit (see Materials and Methods). The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Time-dependence of EPOα-, DarbEPO- and CERA-mediated STAT-5 phosphorylation. HUVECs were treated with medium alone (control) or 1 IU/mL EPOα, 1 IU/mL DarbEPO or 5 IU/mL CERA for different periods of time ranging from 5 to 30 min ( A ). Aliquots of cells were treated with VEGF (25 ng/mL) or granulocyte-macrophage colony-stimulating factor (GM-CSF) (10 IU/mL) for the same time intervals as above ( B ). STAT-5 phosphorylation levels were quantified using an ELISA kit (see Materials and Methods). The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Effect of the STAT-5 inhibitor on EPOα-, DarbEPO- and CERA-mediated STAT-5 phosphorylation. HUVECs were treated with medium alone (basal) or 1 IU/mL EPOα, 1 IU/mL DarbEPO or 5 IU/mL CERA for 5–30 min or VEGF for 10 min in the absence (gray bar) or presence of the STAT-5 inhibitor (80 μM, black bar). STAT-5 phosphorylation levels were quantified using an ELISA kit (see Materials and Methods). The data are expressed as the per cent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments. *** p < 0.001; ** p < 0.01: * p < 0.05 vs . basal value. ### p < 0.001; ## p < 0.01 vs. control (in the absence of inhibitor).

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Effect of erythropoiesis-stimulating agents (ESAs) on HUVEC viability. The cells were treated for 72 h with EPOα (1 IU/mL), DarbEPO (1 IU/mL) or CERA (5 IU/mL) in the absence or presence of 80 μM STAT-5 inhibitor. Cell viability was determined by an MTS assay, as described in the Methods section. The data are expressed as the percent cell viability compared to the untreated basal cells (set to 100%) and represent the mean ± SEM of three different experiments. ** p < 0.01; * p < 0.05 vs. basal value.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: MTS Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: Effect of ESAs on HUVEC angiogenesis. The cells were treated with medium alone (basal) or ESAs ( 1 IU/mL EPOα, 1 IU/mL DarbEPO or 5 IU/mL CERA) for 24 h before seeding onto Matrigel with fresh medium. Capillary-like tube formation was observed by microscopy and quantified using the ImageJ program. ( A ) Representative pictures of HUVEC tubule formation on Matrigel after 24 h drug incubation. ( a ) Basal; ( b ) STAT-5 inhibitor; ( c ) 1 IU/mL EPOα; ( d ) 1 IU/mL DarbEPO; ( e ) 5 IU/mL CERA (original magnification = 10×). ( B ) The number of mesh-like structures were quantified and expressed as a percent of the control sample. The data are expressed as the mean ± SEM of three independent experiments performed in duplicate. *** p < 0.001; ** p < 0.01; * p < 0.05 vs. basal value.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Microscopy, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: EPOR desensitization. ( A ) Time-dependence: cells were pre-treated with medium alone (control) or 1 IU/mL EPOα, 1 IU/mL DarbEPO or 5 IU/mL CERA for different periods of time ranging from 5 to 120 min. ( B ) Concentration-dependence: cells were pre-treated with medium alone (control) or different EPOα, DarbEPO or CERA concentrations (0.5–10 IU/mL) for 60 min. The cells were then washed and stimulated by the addition of 1 IU/mL EPOα, 1 IU/mL DarbEPO or 5 IU/mL CERA for 5 min. STAT-5 phosphorylation levels were quantified using an ELISA kit (see Materials and Methods). The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: EPOR resensitization following 1 IU/mL EPOα- or DarbEPO-induced receptor desensitization. HUVECs were pre-treated with medium alone (control) or 1 IU/mL EPOα ( A ) or 1 IU/mL DarbEPO ( B ) for 18 or 54 min to induce receptor desensitization. Then, cells were the washed and replaced in medium alone for different periods of time (2 to 24 h) to allow for receptor resensitization. All samples were then stimulated by the addition of 1 IU/mL EPOα or 1 IU/mL DarbEPO for 5 min and STAT-5 phosphorylation levels were quantified. The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments. ** p < 0.01; * p < 0.05 vs. DES’.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Regulation of Erythropoietin Receptor Activity in Endothelial Cells by Different Erythropoietin (EPO) Derivatives: An in Vitro Study

doi: 10.3390/ijms14022258

Figure Lengend Snippet: EPOR resensitization following 3 IU/mL EPOα-, 3 IU/mL DarbEPO- or 5 IU/mL CERA-induced receptor desensitization. HUVECs were pre-treated with medium alone (control) or 3 IU/mL EPOα ( A ), 3 IU/mL DarbEPO ( B ) or 5 IU/mL CERA ( C ) for 18 or 54 min to induce receptor desensitization. The cells were then washed and replaced in medium alone for different periods of time (2 to 24 h) to allow for receptor resensitization. All samples were then stimulated by the addition of 3 IU/mL EPOα, 3 IU/mL DarbEPO or 5 IU/mL CERA for 5 min, and STAT-5 phosphorylation levels were quantified. The data are expressed as the percent STAT-5 phosphorylation compared to the untreated control cells (set to 100%) and represent the mean ± SEM of three different experiments. ** p < 0.01; * p < 0.05 vs. DES’.

Article Snippet: The RayBio ® Cell-Based STAT-5 (Tyr694) ELISA kit was purchased from RayBiotech Inc. (Norcross, GA, USA).

Techniques:

Journal: Cellular and Molecular Immunology

Article Title: Chemerin aggravates DSS-induced colitis by suppressing M2 macrophage polarization

doi: 10.1038/cmi.2014.15

Figure Lengend Snippet: Increased production of pro-inflammatory cytokines in chemerin-treated mice following DSS exposure. ( a ) The concentrations of the pro-inflammatory cytokines IL-6, TNF-α and IFN-γ in the supernatant of colon cells after 24 h of culture were measured by ELISA. ( b ) The serum levels of the pro-inflammatory cytokines IL-6 and TNF-α were measured by ELISA. * P <0.05, ** P <0.01, *** P <0.001. The data are expressed as the mean±s.e.m. ( n =4–6 per group). Similar results were obtained in three independent experiments with 4–6 mice per group. DSS, dextran sulfate sodium; ELISA, enzyme-linked immunosorbent assay; IFN, interferon.

Article Snippet: The concentrations of chemerin were measured using an ELISA Kit for mouse chemerin (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Immunology

Article Title: Chemerin aggravates DSS-induced colitis by suppressing M2 macrophage polarization

doi: 10.1038/cmi.2014.15

Figure Lengend Snippet: Attenuated intestinal inflammation in neutralizing anti-chemerin (ChAb)-treated mice following DSS exposure. ( a ) The methods for DSS-induced colitis and ChAb administration. ( b ) Representative photomicrographs of colon sections stained with H&E were examined at ×40 magnification. ( c ) Histological scores were determined as described in the section on ‘Materials and methods'. ( d ) The percentages of neutrophils, DCs and macrophages in the colons were determined by flow cytometry as described above. ( e ) The concentrations of the pro-inflammatory cytokines IL-6, TNF-α and IFN-γ in the supernatant of colon cells after 24 h of culture were measured by ELISA. ( f ) The colonic expression of the M2 macrophage-associated genes Arg1, Ym1, FIZZ1 and IL-10 was examined by RT-PCR analysis. The data are expressed as the mean±s.e.m. ( n =5 per group). Similar results were obtained in three independent experiments with five mice per group. ns=not significant, * P <0.05, ** P <0.01, *** P <0.001. ChAb, chemerin antibody; DC, dendritic cell; DSS, dextran sulfate sodium; ELISA, enzyme-linked immunosorbent assay; IFN, interferon; UC, ulcerative colitis.

Article Snippet: The concentrations of chemerin were measured using an ELISA Kit for mouse chemerin (R&D Systems).

Techniques: Staining, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Oncogene

Article Title: Increased metabolites of 5-lipoxygenase from hypoxic ovarian cancer cells promote tumor-associated macrophage infiltration.

doi: 10.1038/onc.2014.85

Figure Lengend Snippet: Figure 2. Key proteins and metabolites of the 5-LOX pathway are increased in hypoxic ovarian cancer cells. (a) SKOV-3 and OVCAR-3 ovarian cancer cells were treated with 1% O2 for 0, 4, 8, 12 and 24 h. Transcript levels of 5-LOX, FLAP and LTA4H were analyzed by quantitative reverse transcriptase-PCR. *Po0.05 versus 0 h. (b) SKOV-3 cells were treated with 1% O2 for 0, 6, 12, 24 and 48 h. The expression of HIF-1α, 5-LOX and FLAP were analyzed by western blotting. *Po0.05 versus Con. (c) SKOV-3 cells were treated with 1% O2 for 6, 12, 24, 48 and 72 h. CM was harvested and analyzed by ELISA. *Po0.05 versus normoxia. (d) SKOV-3 cells were transfected with siRNAs and treated with 1% O2 for 24 h. The expression of HIF-1α, 5-LOX and FLAP were analyzed by western blotting. *Po0.05 versus Con siRNA. #Po0.05 versus corresponding Con siRNA.

Article Snippet: For measurement of secreted HB-EGF and TNF-α, levels of HB-EGF and TNF-α present in the supernatants were measured using the human HB-EGF ELISA kit (Cusabio Biotech Co., Ltd) and human TNF-α ELISA Kit (R&D Systems) following the manufacture’s protocol.

Techniques: Reverse Transcription, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Transfection

Journal: Oncogene

Article Title: Increased metabolites of 5-lipoxygenase from hypoxic ovarian cancer cells promote tumor-associated macrophage infiltration.

doi: 10.1038/onc.2014.85

Figure Lengend Snippet: Figure 6. Metabolites of 5-LOX enhance release of TNF-α and HB-EGF through upregulation of MMP-7. TNF-α and HB-EGF in medium were detected by ELISA. *Po0.05 versus Con. #Po0.05 versus corresponding CM, CM+isotype-IgG or isotype-IgG. Macrophages were cultured with medium alone (Con), normoxia (N) or hypoxia (H) CM from SKOV-3 cells (a) or untreated and MK886-treated SKOV-3 cells (b). (c) Isotype-IgG or anti-MMP-7 were added in the CM. Macrophages were treated with 5-HETE (d) or LTB4 (e). (f) Macrophages were treated with 5-HETE or LTB4. Then isotype-IgG or anti-MMP-7 were added in the medium.

Article Snippet: For measurement of secreted HB-EGF and TNF-α, levels of HB-EGF and TNF-α present in the supernatants were measured using the human HB-EGF ELISA kit (Cusabio Biotech Co., Ltd) and human TNF-α ELISA Kit (R&D Systems) following the manufacture’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture