Journal: Cells

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

doi: 10.3390/cells11091532

Figure Lengend Snippet: 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/cm 2 . 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: Slides were coated with rmP-Selectin (His-tagged, R&D systems), mouse soluble ICAM1 (STEMCELL Technologies, Vancouver, Kanada), and rmKC (R&D systems) in coating buffer overnight.

Techniques: Generated, Transduction, Expressing, Isolation, 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: 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 expressing 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 −/− macrophages were either kept in suspension or plated on ICAM1 for 20 min to assess integrin-mediated signaling. Western blot analyses for Y402-phosphorylated and total Pyk2; Y31-phosphorylated and total paxillin; T202/Y204-phosphorylated and total ERK1/2; talin; kindlin3; and general tyrosine phosphorylation. GAPDH served as loading control. All values are given as mean ± SD. *** p < 0.001.

Article Snippet: Slides were coated with rmP-Selectin (His-tagged, R&D systems), mouse soluble ICAM1 (STEMCELL Technologies, Vancouver, Kanada), and rmKC (R&D systems) in coating buffer overnight.

Techniques: Expressing, Knock-Out, Derivative Assay, Sequencing, Binding Assay, Western Blot

Journal: Cells

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

doi: 10.3390/cells11091532

Figure Lengend Snippet: 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/cm 2 . 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/cm 2 . 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: Slides were coated with rmP-Selectin (His-tagged, R&D systems), mouse soluble ICAM1 (STEMCELL Technologies, Vancouver, Kanada), and rmKC (R&D systems) in coating buffer overnight.

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