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

Journal: American Journal of Cancer Research

Article Title: LPPR4 promotes peritoneal metastasis via Sp1/integrin α/FAK signaling in gastric cancer

doi:

Figure Lengend Snippet: LPPR4 activates transcription of integrin α through Sp1. A. Classical members of integrin family proteins and corresponding downstream genes were assessed by Western blotting in HGC27 and MKN74 cells transfected with siLPPR4 and in MGC803 cells infected with LPPR4 overexpression plasmid. B. Immunoprecipitation using a LPPR4 antibody showed LPPR4 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 association. C. Correlation between LPPR4 and Sp1, as well as Sp1 and ITGA1, ITGA2, ITGA5, ITGA6, ITGA7 was performed in human GC tissues based on TCGA-STAD dataset. D. Sp1 mRNA expression levels were tested by qRT-PCR in HGC-27 and MKN74 cells after transfected with siLPPR4. E. Sp1 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siLPPR4. F, G. HGC27 and MKN74 cells were transfected with NC-siRNA and Sp1-siRNA. Sp1 expression levels were detected by qRT-PCR and Western blotting. ITGA1, ITGA2, ITGA5, ITGA6 and ITGA7 protein expression levels were detected by Western blotting analysis in HGC27 and MKN74 cells after transfected with siSp1. β-actin was used as a loading control in Western blotting. Each experiment was repeated at least three times. All the data were expressed as mean ± SD, ***P < 0.001, based on Student’s t-test.

Article Snippet: Rabbit anti-Akt (#9272S), anti-phospho-Akt (Ser473) (#9271L), anti-Src (#2110S), anti-phospho-Src (#6943S), anti-FAK (#3285S), anti-phospho-FAK (Y397) (#3281S), anti-integrin β1 (#9699S), anti-integrin β2 (#73663S), anti-integrin β3 (#13166S), anti-integrin α5 (98204S), anti-Sp1 (#9389S) and mouse anti-β-actin (#3700S) were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot, Transfection, Infection, Over Expression, Plasmid Preparation, Immunoprecipitation, Expressing, Quantitative RT-PCR

Journal: Scientific Reports

Article Title: Int6/eIF3e Silencing Promotes Placenta Angiogenesis in a Rat Model of Pre-eclampsia

doi: 10.1038/s41598-018-27296-2

Figure Lengend Snippet: IB4 ( A , B ) cytokeratin immunostaining in close to maternal (a) center (b) and close to the fetal sides (c) of placentas from si-RNA-Int6 and NC-siRNA treated rats. siRNA-Int6 = pre-eclampsia rats treated with siRNA against Int6; NC-siRNA = pre-eclampsia rats treated with siRNA vector negative control.

Article Snippet: Briefly, after deparaffinization, hydration, antigen repairing, decreased endogenous peroxidase activity and blockade with 3% normal bovine serum, the sections were incubated with either CD31 antibodies (Abcam, ab119339, 1:200, Cambridge, MA, USA), Int6 antibodies (Abcam, ab36766, 1:1000, Boston, MA, USA), IB4 antibodies (sc-65254, Santa Cruz Biotechnology, USA.) and basic cytokeratin antibodies (3G129, sc-70402, Santa Cruz Biotechnology, USA.) at 4 °C overnight.

Techniques: Immunostaining, Plasmid Preparation, Negative Control

Journal: Scientific Reports

Article Title: An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding

doi: 10.1038/srep11630

Figure Lengend Snippet: Dissociation constant (K d ) values determined for integrin β2 tail and Dok1 interactions *.

Article Snippet: Cells (2 × 10 6 ) were transfected with integrin αL plasmid (9 μg), integrin β2-YFP plasmid (WT or mutant) (9 μg) and Dok1-CFP or CFP plasmid (12 μg) by electroporation using the Neon electroporation kit (Invitrogen) on a MP-100 pipette-type microporator (Invitrogen).

Techniques:

Journal: Scientific Reports

Article Title: An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding

doi: 10.1038/srep11630

Figure Lengend Snippet: ( A ) K562 cells transfected with the indicated expression plasmids were lysed. Proteins were resolved on 10% SDS-PAGE under reducing conditions and immunoblotted (IB) with either anti-GFP or anti-Dok1 antibodies. ( B ) Transfected K562 cells were subjected to YFP-photobleach FRET analyses. Each data point represents the mean ± S.D. of ≥30 cells analyzed. ( C ) Flow cytometry analyses of K562 stable line expressing Dok1-CFP that were transfected with integrin αLβ2-YFP and CCR5. Wild-type K562 cells were used as the control group. ( D ) FRET analyses of cells in ( C ) that were treated without or with chemokine RANTES (50 ng/ml) for 10 min at 37 °C. 60 and 39 cells were analyzed for conditions without and with RANTES treatment, respectively. Data point represent mean ± S.D. *,p < 0.05, Student’s t test.

Article Snippet: Cells (2 × 10 6 ) were transfected with integrin αL plasmid (9 μg), integrin β2-YFP plasmid (WT or mutant) (9 μg) and Dok1-CFP or CFP plasmid (12 μg) by electroporation using the Neon electroporation kit (Invitrogen) on a MP-100 pipette-type microporator (Invitrogen).

Techniques: Transfection, Expressing, SDS Page, Flow Cytometry

Journal: Scientific Reports

Article Title: An Alternative Phosphorylation Switch in Integrin β2 (CD18) Tail for Dok1 Binding

doi: 10.1038/srep11630

Figure Lengend Snippet: Comparison of amino acid sequences of integrin cytoplasmic tails highlighting the NxxY/F motif (blue) and potential phosphorylation of Ser residues (red).

Article Snippet: Cells (2 × 10 6 ) were transfected with integrin αL plasmid (9 μg), integrin β2-YFP plasmid (WT or mutant) (9 μg) and Dok1-CFP or CFP plasmid (12 μg) by electroporation using the Neon electroporation kit (Invitrogen) on a MP-100 pipette-type microporator (Invitrogen).

Techniques: