Journal: Signal Transduction and Targeted Therapy

Article Title: Carbamylated erythropoietin regulates immune responses and promotes long-term kidney allograft survival through activation of PI3K/AKT signaling

doi: 10.1038/s41392-020-00232-5

Figure Lengend Snippet: Influence of CEPO on DC and CD4 + T cells differentiation and the PI3K/AKT signaling pathway. a Incidence of CEPO on expression of CD11b, CD80, CD86, and MHC-II by DC analyzed by flow cytometry. b Levels of IL-6, IL-10, IL-12, TNF-α, TGF-β, and MCP-1 secreted by the CEPO-treated DC. c qPCR quantitation of mRNA levels of IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-10, IL-13, and IL-17 in CEPO-treated CD4 + T cells. d Western blot analysis of production of IL-2, IFN-γ, TNF-α, Th2, IL-4, IL-5, IL-10, IL-13, and IL-17 by CD4 + T cells. GAPDH was used to normalize each protein expression. e Flow analysis of Th1, Th2, Treg, Th17 cells and the ratio of Th1/Th2, Treg/Th17 cells. f Cell apoptosis was detected by flow cytometry with Annexin V/PI staining. g Western blot analysis of protein levels of p-PI3K, T-PI3K, p-AKT, and T-AKT in DC after CEPO treatment. h Western blot analysis of protein levels of p-PI3K, T-PI3K, p-AKT, and T-AKT in CD4 + T cellsafter CEPO treatment. Images shown are representative of at least three independent experiments, data are expressed as mean ± SEM (* p < 0.05, p values were calculated by Student’s t -test)

Article Snippet: Cultured cells or cells harvested from peripheral blood were stained with fluorochrome-conjugated anti-rat CD4, CD25, CD80, CD86, CD11c, CD11b, IL-4, MHC-II, IFN-γ, and ROR-γt, Foxp3 Abs (BD Biosciences) and analyzed for expression of various cell surface or intracellular markers using a FACSCalibur platform (BD Biosciences).

Techniques: Expressing, Flow Cytometry, Quantitation Assay, Western Blot, Staining

Journal: Signal Transduction and Targeted Therapy

Article Title: Carbamylated erythropoietin regulates immune responses and promotes long-term kidney allograft survival through activation of PI3K/AKT signaling

doi: 10.1038/s41392-020-00232-5

Figure Lengend Snippet: Influence of the PI3K/AKT signaling pathway on expression of pro-inflammatory cytokines determined by adding LY294002 to DC and CD4 + T cells. a Flow cytometric analysis of cell surface CD11b, CD80, CD86 and MHC-II expression by DC after addition of LY294002 ± CEPO. b The influence of LY294002 ± CEPO on the Th1, Th2, and Th1/Th2 ratio was determined by flow cytometry. c qPCR analysis of mRNA expression levels of IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-10, and IL-13 in CD4 + T cells treated with LY294002, CEPO, or LY294002+CEPO, as indicated. d Protein expression of IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-10, and IL-13 in CD4 + T cells treated with LY294002, CEPO, or c LY294002+CEPO was examined by western blot assay. GAPDH was used to normalize each protein expression. Images shown are representative of at least three independent experiments, data are expressed as mean ± SEM (* p < 0.05, p values were calculated by Student’s t -test)

Article Snippet: Cultured cells or cells harvested from peripheral blood were stained with fluorochrome-conjugated anti-rat CD4, CD25, CD80, CD86, CD11c, CD11b, IL-4, MHC-II, IFN-γ, and ROR-γt, Foxp3 Abs (BD Biosciences) and analyzed for expression of various cell surface or intracellular markers using a FACSCalibur platform (BD Biosciences).

Techniques: Expressing, Flow Cytometry, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Carbamylated erythropoietin regulates immune responses and promotes long-term kidney allograft survival through activation of PI3K/AKT signaling

doi: 10.1038/s41392-020-00232-5

Figure Lengend Snippet: Influence of siRNA-PI3K ± CEPO on the phenotype and function of immune cells. a Western blot analysis of the efficiency of PI3K inhibition in DC and CD4 + T cells after 48 h of cell transfection. b Flow cytometric analysis of the percentages of cell population with positive expression for CD11b, CD80, CD86, or MHC-II in DCs treated with siRNA control, siRNA control + CEPO, siRNA-PI3K, or siRNA-PI3K+CEPO, as indicated. c The influence of siRNA-PI3K and CEPO on the Th1, Th2, and Th1/Th2 ratio were determined by flow cytometry. d , e qPCR and western blot analysis of mRNA and protein level expression of IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-10, and IL-13 in CD4 + T cells treated with siRNA control, siRNA control + CEPO, siRNA-PI3K, or siRNA-PI3K+CEPO. GAPDH was used to normalize each protein expression. Images shown are representative of at least three independent experiments, data are expressed as mean ± SEM (* p < 0.05, p values were calculated by Student’s t -test)

Article Snippet: Cultured cells or cells harvested from peripheral blood were stained with fluorochrome-conjugated anti-rat CD4, CD25, CD80, CD86, CD11c, CD11b, IL-4, MHC-II, IFN-γ, and ROR-γt, Foxp3 Abs (BD Biosciences) and analyzed for expression of various cell surface or intracellular markers using a FACSCalibur platform (BD Biosciences).

Techniques: Western Blot, Inhibition, Transfection, Expressing, Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Carbamylated erythropoietin regulates immune responses and promotes long-term kidney allograft survival through activation of PI3K/AKT signaling

doi: 10.1038/s41392-020-00232-5

Figure Lengend Snippet: Analysis of EPO receptor (R) levels and the influence of EPOR blocker on DC and T cells. a Effect of CEOP on EPOR mRNA levels in DC and CD4 + T cells was examined by qPCR analysis. b Effect of CEOP on EPOR protein levels in DC and CD4 + T cells was detected by western blotting. c Western Blot analysis of EPOR protein levels after treatment of cells with the selective PI3K/AKT signaling pathway inhibitor LY294002. d DC cells were treated with CEOP, EPOR blocker, or CEOP+EPOR blocker, as indicated. The expression of CD11b, CD80, CD86, and MHC-II in DC were evaluated by flow cytometric analysis. e qPCR analysis of the mRNA levels of IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-10 and IL-13 in CD4 + T cells treated with the CEOP, EPOR blocker, or CEOP+EPOR blocker, as indicated. f Western Blot analysis of IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-10, and IL-13 protein expression in CD4 + T cells treated with EPOR blocker, EPOR blocker + CEPO, or CEOP alone. g Influence of EPOR blocker, CEPO alone, and EPOR blocker + CEPO on the Th1, Th2, and Th1/Th2 ratio was determined by flow cytometry. Images shown are representative of at least three independent experiments, data are expressed as mean ± SEM (* p < 0.05, p values were calculated by Student’s t -test)

Article Snippet: Cultured cells or cells harvested from peripheral blood were stained with fluorochrome-conjugated anti-rat CD4, CD25, CD80, CD86, CD11c, CD11b, IL-4, MHC-II, IFN-γ, and ROR-γt, Foxp3 Abs (BD Biosciences) and analyzed for expression of various cell surface or intracellular markers using a FACSCalibur platform (BD Biosciences).

Techniques: Western Blot, Expressing, Flow Cytometry

Journal: Signal Transduction and Targeted Therapy

Article Title: Carbamylated erythropoietin regulates immune responses and promotes long-term kidney allograft survival through activation of PI3K/AKT signaling

doi: 10.1038/s41392-020-00232-5

Figure Lengend Snippet: Influence of CEPO, EPOR blocker, and PI3K/AKT inhibitor on Protein levels of EPOR, PI3K, and AKT. a Protein expression of EPOR in DC and CD4 + T cells from renal transplant recipient. b Protein expression of PI3K and AKT in DC from renal transplant recipients. c Protein expression of PI3K and AKT in CD4 + T cells from renal transplant recipients. GAPDH was used to normalize each protein expression. The representative images of each group from 5 samples were shown. Data are expressed as mean ± SEM (* p < 0.05, p values were calculated by Student’s t -test)

Article Snippet: Cultured cells or cells harvested from peripheral blood were stained with fluorochrome-conjugated anti-rat CD4, CD25, CD80, CD86, CD11c, CD11b, IL-4, MHC-II, IFN-γ, and ROR-γt, Foxp3 Abs (BD Biosciences) and analyzed for expression of various cell surface or intracellular markers using a FACSCalibur platform (BD Biosciences).

Techniques: Expressing

Journal: Scientific Reports

Article Title: Histone deacetylase 3 is required for iNKT cell development

doi: 10.1038/s41598-017-06102-5

Figure Lengend Snippet: Hdac3-deficient iNKT cells have a defect in the differentiation of Tbet expressing NKT1. ( a ) Examination of thymic NKT subsets (NKT1, NKT2 and NKT17) as characterized by the expression of Tbet, PLZF and ROR-γt. NKT1 (Tbet + PLZF med ), NKT2 (PLZF hi ROR-γt − Tbet − ) and NKT17 (ROR-γt + PLZF med Tbet − ) in WT and PLZF-cre Hdac3 cKO mice. Data is representative of at least 8 mice per genotype. ( b ) Absolute cell count for NKT2, NKT17 and NKT1 cells from WT (black bar) and PLZF-cre Hdac3 cKO (white bar) mice. Data is calculated from 14 WT and 8 PLZF-cre Hdac3 cKO mice. Statistical analysis was performed using a Student t test. Means ± SEM. ( c ) Production of IL-4 and IL-17A by stimulated (black line) and unstimulated (grey filled) NKT2 and NKT17 cells in the WT and PLZF-cre Hdac3 cKO after 6 hr stimulation with PMA/ionomycin. NKT subsets were defined as described in ( a ). Data is representative of at least 5 mice per genotype from 3 independent experiments. ( d ) Quantification of average MFI of IL-4 and IL-17A production by NKT2 and NKT17, respectively from WT (black bar) and PLZF-cre Hdac3 cKO (white bar). Data is calculated from 5 WT and 5 PLZF-cre Hdac3 cKO mice. Statistical analysis was performed using a Student t test. Means ± SEM. ( e ) Production of IFN-γ by stimulated (black line) and unstimulated (grey filled) NKT1 cells in the WT and PLZF-cre Hdac3 cKO after 6hr stimulation with PMA/ionomycin. NKT subsets were defined as described in ( a ). Data is representative of 5 mice per group. ( f ) Quantification of average MFI of IFN-γ production by NKT1 from WT (black bar) and PLZF-cre Hdac3 cKO (white bar) mice. Data is calculated from 5 WT and 5 PLZF-cre Hdac3 cKO mice. Statistical analysis was performed using a Student t test. Means ± SEM.

Article Snippet: BD bioscience ROR-γt (B2D).

Techniques: Expressing, Cell Counting