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: CytoJournal

Article Title: Association of immunosuppressive CD45 + CD33 + CD14 − CD10 − HLA-DR −/low neutrophils with poor prognosis in patients with lymphoma and their expansion and activation through STAT3/arginase-1 pathway in vitro

doi: 10.25259/Cytojournal_165_2024

Figure Lengend Snippet: List of primer sequences used for RT-PCR analysis.

Article Snippet: After blocking with 5% bovine serum albumin (Gibco, Waltham, MA, USA), the membranes were incubated overnight at 4°C with the following primary antibodies: b-actin (1 μg/mL, Cat.No AF5003, Beyotime, Shanghai, China), STAT1 (2 μg/mL, Cat.No YT4439), STAT3 (2 μg/mL, Cat.No YT4443), STAT5 (2 μg/mL, Cat.No YT4453), phospho-STAT1 (p-STAT1, 2 μg/mL, Cat.No YP0249), phospho-STAT3 (p-STAT3, 2 μg/mL, Cat.No YP0250), and phospho-STAT5 (p-STAT5, 2 μg/mL, Cat.No YP0254) (all antibodies from ImmunoWay Biotechnology, USA, except b-actin).

Techniques:

Journal: CytoJournal

Article Title: Association of immunosuppressive CD45 + CD33 + CD14 − CD10 − HLA-DR −/low neutrophils with poor prognosis in patients with lymphoma and their expansion and activation through STAT3/arginase-1 pathway in vitro

doi: 10.25259/Cytojournal_165_2024

Figure Lengend Snippet: Increased activity of STAT3, which is required for T-cell suppression. (a 1 ) Neutrophils before and after induction were tested for STAT1, STAT3, STAT5, p-STAT1, p-STAT3, and p-STAT5 expression by WB. (a 2 ) Grayscale analysis of WB results by image J software; the result was expressed as the gray value of the target band/the gray value of the reference protein. Increased expression levels of STAT3 and p-STAT3 proteins were found in neutrophils AI compared with those before induction. (b) Changes of mRNA expression of STAT1, STAT3, and STAT5 in neutrophils before and after induction. The mRNA level of STAT3 in neutrophils AI was significantly higher than that in neutrophils before induction. (c 1 and c 2 ) Induced neutrophils with or without ruxolitinib were cocultured or indirectly cocultured with autologous CD3 + T cells from the same donors at 2:1 ratio for 48 h; T-cell proliferation was evaluated by CFSE labeling, and unstimulated T cells were used as a negative control. The samples were analyzed by flow cytometry. Ruxolitinib addition almost completely abolished the ability of CD10 − neutrophils to suppress T-cell proliferation. (c 3 ) Representative flow cytometry data of T-cell proliferation from one individual in coculture and indirect coculture systems. The red area represented the proliferating T-cell fraction. (d 1 and d 2 ) Induced neutrophils with or without ruxolitinib were cocultured or indirectly cocultured with autologous CD3 + T cells from the same donors at 2:1 ratio for 48 h, and apoptotic T cells were assessed by flow cytometry. CD3 + T cells cultured alone were used as a negative control. Ruxolitinib addition almost completely prevented T-cell apoptosis. (d 3 ) Representative flow cytometry data of CD3 + T-cell apoptosis experiment from one individual in the coculture or indirect coculture system. CD3 + T cells without PI or annexin V were used as a blank control. Apoptotic T cells were marked as CD3 + /annexin + /PI + . (e 1 ) WB analysis of the changes in STAT3 and p-STAT3 expression before and after ruxolitinib addition in the induced neutrophil population and (e 2 ) grayscale analysis of WB results by imageJ software. Ruxolitinib inhibited the expression of STAT3 and p-STAT3 proteins. (f) Extracellular production of Arg-1 protein was evaluated by ELISA at 48 h before and after the addition of ruxolitinib in neutrophils AI . Significantly decreased expression of extracellular Arg-1 in neutrophils AI treated with ruxolitinib. (g) Changes in intracellular Arg-1 fluorescence intensity were detected by flow cytometry at 48 h before and after ruxolitinib addition in neutrophils AI . Significantly decreased expression of intracellular Arg-1 in neutrophils AI treated with ruxolitinib, mainly in the CD10 − neutrophil subset. (h) Arg-1 activity was determined at 48 h before and after ruxolitinib addition in neutrophils AI . A significant decrease in Arg-1 activity was observed in the cells treated with ruxolitinib. (i) mRNA expression of Arg-1 was detected by RT-PCR in neutrophils AI at 48 h before and after ruxolitinib addition. A significant decrease was observed in the mRNA expression of Arg-1 in the cells treated with ruxolitinib. (j) mRNA expression of STAT3 was detected by RT-PCR in neutrophils at 48 h before and after ruxolitinib addition. The expression of STAT3 gene in CD10 − neutrophil subpopulation significantly decreased. Each experiment was repeated 3 times. ✶ P < 0.05, ✶ ✶ P < 0.01, ✶ ✶ ✶ P < 0.001, ✶ ✶ ✶ ✶ P < 0.0001, ns: P > 0.05. Ctrl: Neutrophils before induction, neutrophils AI : Neutrophils after induction, WB: Western blot, CFSE: Carboxyfluorescein succinimidyl ester, CD10 − neutrophils: CD45 + CD14 − CD33 + HLA-DR − CD10 − cells, CD10 + neutrophils: CD45 + CD14 − CD33 + HLA-DR − CD10 + cells.

Article Snippet: After blocking with 5% bovine serum albumin (Gibco, Waltham, MA, USA), the membranes were incubated overnight at 4°C with the following primary antibodies: b-actin (1 μg/mL, Cat.No AF5003, Beyotime, Shanghai, China), STAT1 (2 μg/mL, Cat.No YT4439), STAT3 (2 μg/mL, Cat.No YT4443), STAT5 (2 μg/mL, Cat.No YT4453), phospho-STAT1 (p-STAT1, 2 μg/mL, Cat.No YP0249), phospho-STAT3 (p-STAT3, 2 μg/mL, Cat.No YP0250), and phospho-STAT5 (p-STAT5, 2 μg/mL, Cat.No YP0254) (all antibodies from ImmunoWay Biotechnology, USA, except b-actin).

Techniques: Activity Assay, Expressing, Software, Labeling, Negative Control, Flow Cytometry, Cell Culture, Control, Enzyme-linked Immunosorbent Assay, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Western Blot