Journal: American journal of transplantation : official journal of the American Society of Transplantation and the American Society of Transplant Surgeons

Article Title: Primary alloproliferative TH1 response induced by immature plasmacytoid dendritic cells in collaboration with myeloid DCs.

doi: 10.1111/j.1600-6143.2005.01097.x

Figure Lengend Snippet: Figure 4: The phenotype of the responder T-cell population is similar in MDC and 5000PDC+100MDC allostimulatory conditions. After 5 days of culture, the alloproliferating CFSE-low T cells were stained with different mAbs to the indicated markers. (A) The expression of IL2Ra, IL12Rb1 and CD154 on proliferating T cells (gated on R1) is shown. The numbers indicate the percentage of cells in each quadrant. (B) Summary of the markers analyzed and detected (+ve) on responding CFSE-low T cells stimulated by 5000MDC (black bars) or 5000PDC+100MDC (white bars) (mean ± SD, n = 4). As most cells expressing the markers are CFSE low, the proportion is calculated based on the proliferating cells (contained in the R2 regions). (C) The time-dependent expression of IL12Rb2 on CFSE-low T cells was analyzed in three different culture experiments stimulated by (5000PDC+100MDC) (white symbols) or 5000MDCs alone (black symbols) for the indicated time period.

Article Snippet: The following murine monoclonal antibodies (mAbs) were used: Peridinin Chlorophyll Protein (PerCP)-labeled CD3 and HLA-DR; Fluorescein isothiocyanate (FITC)-labeled CD4 and CD45RA; Phycoerythrin (PE)-labeled CD11c, CD123, IL2Ra, CD154, CD45RO, IL12Rb1, IL12Rb2, IL18Ra (BD Biosciences Oxford, UK); TRI-Color® (TC)-labeled CD3, PE-labeled IL4, Allophycocyanin (APC)-labeled IFNc (Caltag Laboratories, Hamburg, Germany); FITC-labeled anti-BDCA2 (Miltenyi Biotec, Bergisch Gladbach, Germany); RPE-Cy5-labeled CD14, CD19 (Serotec Ltd, Kidlington, Oxford, UK).

Techniques: Staining, Expressing

Journal: Diabetes

Article Title: miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

doi: 10.2337/db10-0892

Figure Lengend Snippet: TGF-β2 induces EMT-like changes in proximal tubular epithelial cells. A : NRK52E cells were cultured in the presence of TGF-β2 (10 ng/ml, 3 days). Light microscopy images (×20) demonstrate that TGF-β2 causes a loss of the typical epithelial morphology to larger and more irregular shaped cells typical of the myofibroblast phenotype. B : After treatment with TGF-β2 (10 ng/ml, 3 days), gene expression levels were assessed by real-time QPCR. A significant increase was observed in the expression of profibrotic factors (TGF-β1, CTGF), fibrotic genes (αSMA, fibronectin [Fib], collagen [Col] I and IV), and PAI-1, but E-cadherin (E-cad) was significantly reduced (* P < 0.05 compared with control). C : Western analysis demonstrated that αSMA and Col I were both significantly elevated at the protein level after TGF-β2 treatment, while E-cadherin was decreased. D : The results from the Western analysis in C are shown as a graph (* P < 0.05 compared with control).

Article Snippet: Recombinant human TGF-β1, TGF-β2, normal goat IgG, and TGF-β2 neutralizing antibody were from R&D systems (Minneapolis, MN) and used at specified concentrations.

Techniques: Cell Culture, Light Microscopy, Expressing, Western Blot

Journal: Diabetes

Article Title: miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

doi: 10.2337/db10-0892

Figure Lengend Snippet: TGF-β1-induced ECM gene and TGF-β2 expression changes in proximal tubular epithelial cells. A : NRK52E cells (Dulbecco's modified Eagle medium, 25 mmol/l glucose, 2% serum) were treated with TGF-β1 (10 ng/ml, 3 days), and the expression of several genes was assessed by real-time QPCR. Significant changes are indicated (* P < 0.05 compared with control). B : The change in expression of TGF-β1 and TGF-β2 genes was assessed by real-time QPCR, and significant changes are indicated (* P < 0.0005 compared with control). C : TGF-β2 proteins levels were measured by ELISA and expressed as pg/mg (* P < 0.0005 compared with control). D : NRK52E cells were incubated with either control IgG (1 μg/ml) or TGF-β2-specific neutralizing antibody (1 μg/ml) for 1 h, and then TGF-β1 was added (10 ng/ml). Cells were harvested 3 days later and subjected to real-time QPCR analysis. The TGF-β2 antibody prevented the increased expression of αSMA, Collagen I, and fibronectin that is induced by TGF-β1, compared with the IgG control antibody (* P < 0.05 compared with control). E : Cells were then treated with TGF-β1 (10 ng/ml, 3 days) in the presence or absence of SB432542, the TGF-β2 receptor inhibitor. Real-time QPCR analysis confirmed that the gene expression changes induced by TGF-β1 are attenuated by SB432542 (* P < 0.05 and # P < 0.001 compared with control).

Article Snippet: Recombinant human TGF-β1, TGF-β2, normal goat IgG, and TGF-β2 neutralizing antibody were from R&D systems (Minneapolis, MN) and used at specified concentrations.

Techniques: Expressing, Modification, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Diabetes

Article Title: miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

doi: 10.2337/db10-0892

Figure Lengend Snippet: TGF-β1-induced changes in miRNA expression. A : NRK52E cells were cultured in the presence of TGF-β1 (10 ng/ml, 3 days) before miRNA expression levels were assessed by real-time QPCR. A significant decrease was observed in miR-200a but not in miR-141 (* P < 0.03 compared with control). B : SB432542 also attenuated the TGF-β1-induced reduction of miR-200a (* P < 0.02 compared with control). C : Treatment of NRK52E cells with TGF-β2 (10 ng/ml, 3 days) also caused reduction in the expression of miR-200a, miR-200b, and miR-200c (* P < 0.05 compared with miR-control).

Article Snippet: Recombinant human TGF-β1, TGF-β2, normal goat IgG, and TGF-β2 neutralizing antibody were from R&D systems (Minneapolis, MN) and used at specified concentrations.

Techniques: Expressing, Cell Culture

Journal: Diabetes

Article Title: miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

doi: 10.2337/db10-0892

Figure Lengend Snippet: miR-200a represses the expression of ECM proteins. A : NRK52E cells were transfected with miR-200a (100 nmol/l), and RNA was harvested after 3 days for real-time QPCR analysis. miR-200a resulted in significantly decreased expression of several ECM proteins including αSMA, collagen (Col) I and IV, and fibronectin (Fibr) (* P < 0.05 compared with control transfected cells). Expression of TGF-β2 was also significantly decreased as was PAI-1, which is downstream of the TGF-β signaling pathway. The expression of E-cadherin (E-cad) was significantly elevated. B : Western analysis demonstrated a significant decrease in αSMA and collagen I by miR-200a, consistent with the RNA expression analysis (* P < 0.05 compared with control transfected cells). The Westerns were quantified and also shown in graph format below the Western blots (* P < 0.05 compared with control). C : NRK52E cells were cotransfected with the p(CAGA) 12 SMAD3 activity reporter construct, a β-galactosidase construct, and miR-200a. Four h later, the cells were treated with TGF-β1, and cells were harvested after 3 days. TGF-β1 resulted in increased SMAD3 activity with miR-C, which was strongly inhibited by miR-200a (* P < 0.00005 compared with miR-C control; # P < 0.0005 compared with miR-C with TGF-β1; $ P < 0.002 compared with miR-200a control). D : Western analysis of phospho-SMAD3 and total SMAD3 levels in miR-200a-transfected NRK52E demonstrating reduced SMAD3 phosphorylation relative to total SMAD3 protein (* P < 0.05 compared with miR-C control). The Westerns were quantified and shown in graph format (* P < 0.05 compared with miR-C).

Article Snippet: Recombinant human TGF-β1, TGF-β2, normal goat IgG, and TGF-β2 neutralizing antibody were from R&D systems (Minneapolis, MN) and used at specified concentrations.

Techniques: Expressing, Transfection, Western Blot, RNA Expression, Activity Assay, Construct

Journal: Diabetes

Article Title: miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

doi: 10.2337/db10-0892

Figure Lengend Snippet: miR-141a shares the same seed sequence with miR-200a and represses the expression of ECM proteins. A : Alignment of the miR-141/200a sequences and the targeted area of the 3′UTR of TGF-β2 ( http://www.targetscan.org ). Also shown is the altered sequence of the mutant 3′UTR of TGF-β2. B : Proximal tubular cells were transfected with either miR-control or miR-141 (100 nmol/l), and the expression of certain genes was assessed by real-time QPCR. As with miR-200a, miR-141 was able to significantly reduce the expression of αSMA, fibronectin (Fibr), and collagen I (Col I) and resulted in increased expression of E-cadherin (E-cad) (* P < 0.05 compared with control). The expression of TGF-β2 was also significantly reduced.

Article Snippet: Recombinant human TGF-β1, TGF-β2, normal goat IgG, and TGF-β2 neutralizing antibody were from R&D systems (Minneapolis, MN) and used at specified concentrations.

Techniques: Sequencing, Expressing, Mutagenesis, Transfection

Journal: Diabetes

Article Title: miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

doi: 10.2337/db10-0892

Figure Lengend Snippet: The TGF-β2 3′UTR is regulated by miR-141/200a. A : NRK52E cells were transfected with TGF-β2 3′UTR luciferase reporter plasmid (1 μg), β-galactosidase plasmid (0.2 μg), and either miR-control (miR-C), miR-141, or miR-200a (100 nmol/l), and cells were analyzed for β-galactosidase and luciferase activity after 3 days. Both miR-141 and miR-200a were able to significantly repress luciferase activity from the TGF-β2 3′UTR (* P < 0.05 compared with control transfected cells). B : No activity of miR-141 and miR-200a against the mutant TGF-β2 3′UTR was observed. C : miR-141 and ( D ) miR-200a were able to prevent the increased luciferase activity induced by TGF-β1 on the TGF-β2 3′UTR. E : Total TGF-β2 was significantly decreased at the protein level as measured by ELISA and ( F ) at the mRNA level as measured by QPCR in NRK52E cells, 3 days after transfection with miR-200a (* P < 0.05, compared with control).

Article Snippet: Recombinant human TGF-β1, TGF-β2, normal goat IgG, and TGF-β2 neutralizing antibody were from R&D systems (Minneapolis, MN) and used at specified concentrations.

Techniques: Transfection, Luciferase, Plasmid Preparation, Activity Assay, Mutagenesis, Enzyme-linked Immunosorbent Assay

Journal: Diabetes

Article Title: miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

doi: 10.2337/db10-0892

Figure Lengend Snippet: Changes in gene and miR-200a expression in diabetic mouse kidney cortex. A : Immunohistochemical analysis demonstrated increased expression for αSMA, collagen IV (Col IV), and fibronectin (Fibr) in the diabetic mouse kidney cortex compared with control. B : mRNA was extracted from the renal cortex of control and 10-week diabetic apoE mice ( n = 8 per group). Gene expression was assessed by real-time QPCR for a number of genes, revealing significantly increased expression of αSMA, fibronectin, and collagen IV at the RNA level (* P < 0.01 and # P < 0.05 compared with control). C : Expressions of TGF-β1 and TGF-β2 were also elevated at the mRNA level in diabetic mouse kidney cortex (* P < 0.05 and # P < 0.01 compared with control). D : The increased expressions of TGF-β1 and TGF-β2 were associated with decreased expressions of miR-141 and miR-200a in diabetic kidney (* P < 0.05 compared with control). E : Relative expression levels of miR-141 and miR-200a in NRK52E cells and mouse kidney cortex. (* P < 0.005 compared with control). (A high-quality color representation of this figure is available in the online issue.)

Article Snippet: Recombinant human TGF-β1, TGF-β2, normal goat IgG, and TGF-β2 neutralizing antibody were from R&D systems (Minneapolis, MN) and used at specified concentrations.

Techniques: Expressing, Immunohistochemical staining

Journal: Diabetes

Article Title: miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

doi: 10.2337/db10-0892

Figure Lengend Snippet: Changes in miR-141/200a expression in the kidney in the adenine-induced renal fibrosis model. A : Trichrome staining of tissue sections from renal cortex from control and adenine-fed C57bl6 mice after 4 weeks of treatment. Blue staining indicates high levels of collagen in the adenine-fed mouse kidney compared with control. B : mRNA was extracted from the renal cortex of control and adenine-fed C57bl6 mice ( n = 4 per group). Gene expression was assessed by real-time QPCR, revealing significantly increased expression of collagen I, collagen III, fibronectin, vimentin, TGF-β1, and TGF-β2. C : The increased expression of TGF-β1, TGF-β2, and collagen was associated with decreased expression of miR-141 and miR-200a but not the appropriate control, RNU6B, in kidney cortex (* P < 0.05 compared with control). (A high-quality color representation of this figure is available in the online issue.)

Article Snippet: Recombinant human TGF-β1, TGF-β2, normal goat IgG, and TGF-β2 neutralizing antibody were from R&D systems (Minneapolis, MN) and used at specified concentrations.

Techniques: Expressing, Staining

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 1. Integrin β2 can be substituted by its human homolog in mouse Hoxb8 FL cells. (A) Scheme of workflow. Hoxb8 FL cells were generated from bone marrow of integrin β2-deficient mice and retrovirally transduced with mouse or human integrin β2. Human/mouse integrin β2-positive cells were FACS-sorted. (B) Surface expression of different integrin subunits and cell markers on neutrophil-like cells differentiated from control (β2+/+), integrin β2 ko (β2−/−), and human or mouse integrin β2-rescued integrin β2−/−(β2−/−/hβ2 and β2−/−/mβ2) Hoxb8 FL cells assessed by FACS analysis. (C) Integrin β2 surface expression levels on neutrophil-like cells differentiated from integrin β2−/−Hoxb8 FL cells retrovirally transduced with human integrin β2 (β2−/−/hβ2) compared to PMNs isolated from human blood. (D) Static adhesion of untreated, TNFα-treated, or PMA-treated Hoxb8 FL-derived β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 neutrophils on ICAM1. N = 4. Individual data points of the 4 independent experiments are shown. (E,F) Adhesion (E) and rolling velocities (F) of neutrophil-like cells differentiated from β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 Hoxb8 FL cells in flow chambers coated with ICAM1 and P-selectin with or without CXCL1 under constant shear rate of 1 dyn/cm2. N = 10/11 chambers with CXCL1 (rolling and adhesion); N = 3/4 without CXCL1 (adhesion). (G) Neutrophil-like β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 cells plated on a P-selectin-, ICAM1-, and CXCL1-coated surface for 10 min. Scale bar: 100 µm. All values are given as mean ± SD. * p < 0.05, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Generated, Transduction, Expressing, Control, Isolation, Derivative Assay, Shear

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 2. Expression of human integrin β2 rescues spreading and adhesion defects in mouse integrin β2 knockout macrophages. (A) Adhesion of integrin β2−/−and human or mouse integrin β2 ex- pressing integrin β2−/−macrophages to ICAM1 and fibronectin in relation to control macrophages. N = 5. Individual data points represent the 5 independent experiments. (B) Spreading of control, integrin β2−/−, and human or mouse integrin β2 expressing integrin β2−/−macrophages to ICAM1 and fibronectin assessed 2 h after plating. N = 3 independent experiments, shown as individual data points. (C) Hoxb8-derived β2+/+, β2−/−, β2−/−/hβ2, and β2−/−/mβ2 macrophages plated on an ICAM1- or fibronectin-coated surface for 2 h. Scale bar: 100 µm. (D) Alignment of the amino acid sequence of the mouse and human β2 integrin cytoplasmic tails. Talin-binding NPLF and kindlin- binding NPKF motives are highlighted in red. MP, membrane proximal; MD, membrane distal. (E) Control, integrin β2−/−, and human or mouse integrin β2 expressing integrin β2−/−

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Expressing, Knock-Out, Control, Derivative Assay, Sequencing, Binding Assay, Membrane

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 3. Expression of human integrin β2 in mouse integrin β2−/−Hoxb8 cells allows assessment of integrin activity using conformation-specific anti-human integrin β2 antibodies. (A,B) FACS analyses of integrin β2 activation of Hoxb8-derived neutrophils expressing human or mouse integrin β2 by measuring staining intensities of conformation-specific antibodies mAb24 (A) and KIM127 (B) either untreated, EDTA-treated, or in response to TNFα, fMLP, or PMA. (C,D) Relative mAb24 (C) and KIM127 (D) binding to resting or EDTA-, TNFα-, fMLP-, or PMA-stimulated Hoxb8-derived neutrophil-like cells upon treatment with DMSO, the phosphatidylinositol-3-kinase (PI3K) inhibitor Wortmannin, the phospholipase C (PLC) inhibitor U-73122, or the protein kinase C (PKC) inhibitor Gö6983. N = 7 (mAb24) or 5 (KIM127) independent experiments, shown as individual data points. All values are given as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Expressing, Activity Assay, Activation Assay, Derivative Assay, Staining, Binding Assay

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 4. Both talin1 and kindlin3 are required for β2 integrin activation. (A) Scheme of work- flow. Talin1, kindlin3, and talin1/kindlin3 double-deficient Hoxb8 FL cells were generated via the CRISPR/Cas9 system. Single cell clones were screened for talin1 and kindlin3 expression. Four clones per genotype were subjected to CRISPR/Cas9-mediated integrin β2 ablation and retrovirally trans- duced to express human integrin β2. Mouse integrin β2-negative and human integrin β2-positive cells were FACS-sorted. (B) Western blot analysis of neutrophil-like cells differentiated from different human integrin β2 expressing Hoxb8 single cell clones, in which talin1 and/or kindlin3 were ablated with the CRISPR/Cas9 system. GAPDH served as loading control. (C,D) Relative mAb24 (C) and KIM127 (D) binding to neutrophil-like cells derived from Hoxb8 single cell clones, expressing human integrin β2 and lacking talin1 and/or kindlin3 expression. N = 4 clones. Result of each clone is plotted as individual data point. All values are given as mean ± SD. ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Activation Assay, Generated, CRISPR, Clone Assay, Expressing, Western Blot, Control, Binding Assay, Derivative Assay

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 5. Kindlin3 is dispensable for P-selectin-induced integrin αLβ2-mediated slow rolling but required for chemokine-induced slower rolling. (A–C) Adhesion (A), rolling velocities (B), and rolling velocities after LFA-1 blocking (C) of Hoxb8 cell-derived PMN-LCs in flow chambers coated with ICAM1, P-selectin, and CXCL1 under constant shear rate of 1 dyn/cm2. N = 12–16 (A,B) and 6–9 (C) flow chambers. (D) Rolling velocities of PMN-LCs on ICAM1- and P-selectin-coated surfaces (without CXCL1) under constant shear rate of 1 dyn/cm2. N = 8–9 flow chambers. Cells were differentiated from different single cell clones, in which talin1 and/or kindlin3 were ablated with the CRISPR/Cas9 system. Rolling velocities are shown as cumulative distribution of the velocities of approximately 500 (B), 300 (C), and 400 (D) cells. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Blocking Assay, Derivative Assay, Shear, Clone Assay, CRISPR

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 6. Talin and/or kindlin3 binding-deficient human integrin β2 exhibit impaired integrin ac- tivation. (A) Scheme of the integrin β2 cytoplasmic domain, which binds talin1 via a membrane proximal NPLF motif and kindlin3 via a membrane-distal NPKF and a TTT motif (left). Muta- tion of these motifs to NPLA and AAA prevent talin1 and kindlin3 binding, respectively (right). (B) Relative mAb24 binding to neutrophil-like cells derived from Hoxb8 cells expressing human wild-type integrin β2, mutant integrin β2 F/A (F754A), mutant integrin β2 TTT/AAA (T758A, T759A, T760A), or double-mutant integrin F/A TTT/AAA in response to TNFα, CXCL1, fMLP, and PMA or left untreated. N = 5 experiments indicated as individual data points. All values are given as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: Binding Assay, Membrane, Derivative Assay, Expressing, Mutagenesis

Journal: Cells

Article Title: Humanized β2 Integrin-Expressing Hoxb8 Cells Serve as Model to Study Integrin Activation.

doi: 10.3390/cells11091532

Figure Lengend Snippet: Figure 7. Rapid CRISPR-based screening approach to study putative β2 integrin regulators. (A) Scheme

Article Snippet: Mouse or human integrin β2 cDNA were purchased from Addgene (Watertown, MA, USA) and subcloned into pMIGR via XhoI and SalI restriction sites.

Techniques: CRISPR