Journal: Stem Cell Reports

Article Title: A Scalable and Efficient Bioprocess for Manufacturing Human Pluripotent Stem Cell-Derived Endothelial Cells

doi: 10.1016/j.stemcr.2018.07.001

Figure Lengend Snippet: Functional Comparison of 2D-ECs and 3D-ECs (A) Western blotting shows that 3D-ECs have higher expression of CD31, CD144, FN, NOTCH4, and ITGA2 than 2D-ECs. (B and C) Immunostaining (B) and flow cytometry (C) of Ki67 shows more proliferating cells in 2D-ECs than in 3D-ECs. Scale bars, 50 μm. (D) Glycolysis analysis shows that 3D-ECs produce more L-lactates than 2D-ECs. Data are represented as means ± SD of three independent replicates (n = 3). ∗∗∗ p < 0.001.

Article Snippet: Magnetic beads coated with anti-CD144 antibodies (Miltenyi Biotec, cat. no. 130-097-857) were added to pull down ECs with a magnetic cell separator.

Techniques: Functional Assay, Comparison, Western Blot, Expressing, Immunostaining, Flow Cytometry

Journal: Stem Cell Reports

Article Title: A Scalable and Efficient Bioprocess for Manufacturing Human Pluripotent Stem Cell-Derived Endothelial Cells

doi: 10.1016/j.stemcr.2018.07.001

Figure Lengend Snippet: A Prototype Bioreactor (A) The bioreactor consists of a pump for medium perfusion, an oxygen-permeable plastic bag for stocking medium and a closed container (e.g., a 50-mL conical tube), where hydrogel fibers with cells are suspended. (B) On day 0, single hPSCs are mixed with 10% PNIPAAm-PEG solution at 4°C and injected into the room-temperature E8 medium in the container. Fibrous hydrogels are instantly formed. Cells are cultured in E8 medium for 5 days, followed by an additional 5 days in EC differentiation medium. Medium is continuously perfused. On day 10, the hydrogel scaffold is liquefied, and spheroids are pelleted by centrifugation. Spheroids are dissociated into single cells by incubating in Accutase at 37°C for 10 min. Magnetic beads coated with anti-CD144 were added to pull down the CD144+ cells. (C–E) Live/dead staining (C), immunostaining (D), and flow cytometry analysis (E) of day 10 cells. Scale bars, 200 and 100 μm. (F) When transplanted subcutaneously with a Matrigel matrix, ECs form nice vascular structures. H9s are used in this figure. Scale bar, 50 μm.

Article Snippet: Magnetic beads coated with anti-CD144 antibodies (Miltenyi Biotec, cat. no. 130-097-857) were added to pull down ECs with a magnetic cell separator.

Techniques: Injection, Cell Culture, Centrifugation, Magnetic Beads, Staining, Immunostaining, Flow Cytometry

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 1. Trio promotes endothelial barrier function. (A) Endothelial cells are transfected with a TagRFP-labeled control shRNA (TagRFP– shCTRL) or a TagRFP-labeled shRNA against Trio (TagRFP-shTrio) (red) and VE-cadherin– GFP (green). The dynamics of cell–cell junctions were followed over time as indicated (min). Regions of interest (ROI) show VE-cadherin–GFP distribution over time. (B, left) HUVECs were transduced with shCTRL or shTrio and stained as indicated. (Middle) Western blots show Trio knockdown. (Right) The FAJ length versus the total junction length was quantified. Scale bars: 20 µm. (C) Endothelial cells were transduced with control or two different shRNAs against Trio, and electrical resistance was monitored by using ECIS. The bar graph represents electrical resistance one day after seeding. The western blot shows Trio knockdown. (D) Overview of GFP–Trio constructs – full-length Trio (GFP–TrioFL), the N-terminus of Trio containing GEF1 (GFP–TrioN) and the C-terminus containing GEF2 (GFP–TrioC). (E) Endothelial cells were transduced with Trio or control shRNA, followed after 2 days by infection with adenovirus expressing GFP or GFP–TrioN. The bar graph represents the electrical resistance. (F) Endothelial cells were transduced with adenovirus encoding GFP–TrioN or GFP–TrioC. (G) Endothelial cells were transduced with adenovirus encoding GFP–TrioN. Rac1 activity was inhibited by using 50 mM EHT-1864. (H) Endothelial cells expressing GFP or GFP– TrioN were grown to confluence on electrode arrays. One day after cell seeding, a non-blocking (clone 7H1) or a VE-cadherin-blocking (clone 75) antibody was added (6.25 µg/ml). All experiments were performed at least three times. Data are mean±s.e.m. *P<0.05; **P<0.01.

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Transfection, Labeling, Control, shRNA, Transduction, Staining, Western Blot, Knockdown, Construct, Infection, Expressing, Activity Assay, Blocking Assay

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 2. Trio is required for efficient cell–cell junction recovery. (A) Still images and ROIs from time-lapse recordings (supplementary material Movie 3) showing linear stable cell–cell junctions (arrowheads) in GFP– TrioN-expressing cells, 10–30 min after stimulation with thrombin, whereas a large proportion of cell–cell junctions in non-transfected cells are disrupted (asterisks). VE-cadherin is visualized using an AlexaFluor-647-conjugated antibody. (B) Endothelial cells were transfected with control shRNA (shCtrl, black line) or shRNAs against Trio (shTrio, dark blue line) and grown to confluence on fibronectin-coated electrode arrays. At time point 0, cells were incubated with (dashed line) or without (solid line) thrombin. Resistance was monitored over time by ECIS. Arrow indicates the starting point of the recovery phase. The bar graph represents the percentage recovery of the endothelial monolayer resistance after thrombin at time points when control monolayers had been completely restored. (C) Still images of the time-lapse recording (at the indicated times, min) of thrombin-stimulated control or Trio- depleted endothelial cells expressing VE-cadherin– GFP. See also the corresponding supplementary material Movie 4, which is representative of multiple experiments. Arrows indicate the formation of cell–cell junctions during the recovery phase, indicated by arrows in the panels on the right, and white lines indicate gaps appearing in Trio-deficient cells. Analysis of interendothelial gaps based on DIC imaging showed increased gaps in Trio-deficient cells after treatment with thrombin. All experiments were repeated three times. Data are mean± s.e.m. *P<0.05. Scale bars: 20 µm (A); 10 µm (C).

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Stable Transfection, Expressing, Transfection, Control, shRNA, Incubation, Imaging

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 3. Trio localizes at endothelial cell–cell contacts. (A) Endothelial cells were transfected with GFP–TrioFL, GFP–TrioN or GFP–TrioC and stained as indicated. ROIs show colocalization between Trio and VE-cadherin. The profile shows the fluorescence intensity of VE-cadherin (red) and GFP proteins (green) according to the line present in the ROI. (B) CHO cells were transfected with Myc-TrioFL and transduced with adenovirus containing VE-cadherin–GFP. Cells were stained as indicated. ROIs (enlarged in the rectangular panels) show that Myc–TrioFL localization at cell–cell contacts depends on VE-cadherin expression. Arrowheads indicate cell–cell contact areas. (C) Endothelial cells were silenced for VE-cadherin and transfected with GFP–TrioFL, and then stained as indicated. ROIs show no localization of Trio at VE-cadherin-deficient cell–cell contacts, but do show β-catenin. Arrowheads indicate cell–cell contact areas. Scale bars: 20 μm.

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Transfection, Staining, Fluorescence, Transduction, Expressing

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 4. Interaction of Triowith VE-cadherin. (A) Trio immunoprecipitation (IP) from endothelial cell lysates were analyzed by western blotting. VE-cadherin and the catenins were precipitated, whereas N-cadherin, PECAM-1, VEGFR2 and ZO-1 were not. (B) Overview of VE-cadherin constructs. VEΔβ–GFP, deletion of β-catenin-binding domain; VEΔC-α–GFP, cytoplasmic domain is replaced with α-catenin; VEΔC-αΔN–GFP, cytoplasmic domain is replaced with α-catenin lacking the N-terminal β-catenin-binding domain. (C) Cos7 cells were transfected with Myc-tagged TrioFL, and wild-type VE-cadherin–GFP (VE–GFP) or a VE-cadherin mutant, as indicated. VE-cadherin–GFP was immunoprecipitated by using an antibody against GFP (IP GFP), and the binding of Myc–TrioFL was determined by western blotting. The panel on the right shows the quantification of three independent experiments and the fold-change in binding of VE mutants to Trio compared with VE–wt-binding to Trio. Data are mean±s.e.m. HC, heavy chain; LC, light chain; TCL, total cell lysate. (D) Illustration of the VE-cadherin peptides #1 and #2 used. BD, binding domain; Cat, catenin. (E) HEK293 cells were transfected with GFP–TrioFL and lysed. Specific biotin-tagged peptides, encoding regions of the VE-cadherin cytoplasmic tail as indicated, were used to pull down (PD) GFP–Trio. VE-cadherin peptide (VE peptide) #2 efficiently precipitated TrioFL as well as β-catenin. (F) HUVECs were transfected with GFP–Trio-mutants as indicated, and VE-cadherin (VE-Cad) was immunoprecipitated. Western blot shows interaction of VE-cadherin with TrioN but not with GEF1, GEF2 or GFP. Panels on the right show protein expression in total cell lysates (TCL). (G) VE-cadherin peptide #2 was co-incubated with GST-tagged spectrin repeats (Spec.) as indicated. Western blot analysis shows that Trio spectrin repeats 5–6 interacted with VE peptide #2 and not with the scrambled peptide. Experiments were performed three times independently.

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Immunoprecipitation, Western Blot, Construct, Binding Assay, Transfection, Mutagenesis, Expressing, Incubation

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 5. Dynamic interaction of Trio–VE-cadherin. (A) Endothelial cells at different confluences (days after seeding are indicated above) were lysed and subjected to immunoprecipitation (IP) of Trio. Association of VE-cadherin and β-catenin with Trio was determined by western blotting. Quantification is shown in the right panel. TCL, total cell lysate. (B) Cells that had been cultured for 6 days, reaching full confluency, were subjected to Ca2+ switch – treatment with EGTA treatment to chelate extracellular Ca2+ leading to cell–cell junction disruption, followed by EGTA washout and Ca2+

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Immunoprecipitation, Western Blot, Cell Culture, Disruption

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 6. VE-cadherin-induced Rac1 activation depends on Trio. (A–D) VE-cadherin- ectodomain-Fc- or Fc-coated magnetic beads were added to an endothelial monolayer to induce VE-cadherin ligation. (A) Rac1 activation increases 15–30 min after adding VE-cadherin- coated beads, as analyzed using a CRIB-peptide pulldown (PD) assay. Right panel shows the quantification. (B) Endothelial cells were transduced with a control shRNA (shCtrl) or a shRNA against Trio (shTrio #2). VE-cadherin ligation did not increase Rac1 activation in Trio- deficient cells. Right panel shows quantification. Time (min) after addition of VE-cadherin- ectodomain-Fc-coated beads is shown. (C) VE- cadherin ligation was induced in endothelial cells treated with DMSO or the Trio-GEF1 inhibitor ITX3. Treatment with ITX3 blocks VE-cadherin ligation-induced Rac1 activation. Right panel shows quantification. Time (min) after addition of VE-cadherin-ectodomain-Fc-coated beads is shown. (D) Trio-deficient endothelial cells (shTrio) were transfected with GFP, wild-type GFP–TrioN or a mutant GFP–TrioN construct comprising two mutations (N1406A/D1407A, GFP–TrioN-Mut), and the length of the FAJs was quantified as described previously. For each condition, 25 cells were analyzed. All experiments were performed at least three times independently. Data are mean±s.e.m. *P<0.05.

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activation Assay, Magnetic Beads, Ligation, Transduction, Control, shRNA, Transfection, Mutagenesis, Construct

Journal: Journal of cell science

Article Title: A local VE-cadherin and Trio-based signaling complex stabilizes endothelial junctions through Rac1.

doi: 10.1242/jcs.168674

Figure Lengend Snippet: Fig. 7. Spatio-temporal Rac1 activity. (A) Endothelial cells were transfected with the DORA Rac1 biosensor and α-catenin– mCherry to mark cell–cell junctions. Panels show differential interference contrast (DIC) microscopy images, ratiometric images with warm colors as increased FRET (Venus and Cerulean3) signals [see look-up table (LUT) on the right], α-catenin–mCherry and the merge with FRET in red, and the merge with α-catenin–mCherry in white. Arrowheads show colocalization of local active Rac1 with α-catenin. Asterisks show formation of nascent cell–cell junctions. The times after beginning observation are shown. (B) Trio- deficient endothelial cells are marked by TagRFP (TagRFP-shTrio); the junction region is marked by the VE-cadherin-AlexaFluor-647 antibody, because the red channel is used to detect TagRFP. All fluorescent signals were recorded in real time. (C) Quantification of the ratiometric changes at regions of nascent cell–cell junctions, marked by α-catenin or VE-cadherin (dotted line in panels A and D), show an increased FRET signal after approximately 15 min in shCTRL but not in Trio-deficient endothelial cells. The graph shows data that is representative of three independent experiments. Data are mean± s.e.m. (D) Trio-deficient endothelial cells (marked by TagRFP–shTrio) show no increase in FRET signal at sites of newly formed cell–cell junctions, marked by the VE-cadherin-AlexaFluor-647 antibody (arrowheads). Note that the basal FRET signals (LUT) are higher in Trio-deficient cells than in control cells (compare with LUT in A), in line with the biochemical data. Scale bars: 10 μm (A,B); 5 μm (D).

Article Snippet: Monoclonal antibodies (mAb) to β-catenin, p120-catenin, γ-catenin, Cdc42 (clone 44), Rac1, VE-cadherin (clone 75; used at 6.25 μg/ml) and an AlexaFluor-647-conjugated antibody against VE-cadherin (clone 7H1) were purchased from BD Transduction Laboratories (Amsterdam, The Netherlands). mAbs to VE-cadherin (clone F8), RhoA and polyclonal Abs (pAb) to β-catenin, α-catenin and Trio (clone D-20) were purchased from Santa Cruz Biotechnology (Heidelberg, Germany).

Techniques: Activity Assay, Transfection, Microscopy, Control

Journal: Nature Communications

Article Title: Targeting QKI-7 in vivo restores endothelial cell function in diabetes

doi: 10.1038/s41467-020-17468-y

Figure Lengend Snippet: QKI-7 overexpression ( p value: 0.0006) in miPS-ECs led to downregulation of EC function-related genes CD144, TSG6, and NLGN1 ( p values: 0.0027, 0.0012, 0.0014) ( a , b ). Protein level alteration of QKI-7 and target candidates was verified by western blot from three independent experiments ( c ) and ELISA ( p value: 0.0038) ( d ). QKI-7 overexpressing miPS-ECs showed decreased cell index with xCELLigence real-time cell analysis (RTCA) indicating disrupted cell barrier and increased permeability ( p value: 0.0073) ( e ). In tube formation assay, QKI-7 overexpressing miPS-ECs formed less capillary structure than control cells, measured by shorter tube branch length and smaller meshed area ( p values: 0.0069, 0.0024) ( f ). QKI-7 overexpression enhanced THP-1 adhesion to miPS-ECs ( p value: 0.0107) ( g ), the data were normalized by setting the control group as 1. Data are from three biologically independent experiments. Error bars represent mean ± SEM ( n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed t test). The source data are provided as a Source data file.

Article Snippet: Primary antibodies include QKI-7 (UC Davis/NIH NeuroMab Facility 73-200, WB 1:1000, ICC 1:100), CD144 (St John’s Laboratory STJ96234, WB 1:1000, ICC 1:200), CD31 (Abcam AB28364, WB 1:1000, ICC 1:20), KDR (R&D Systems MAB3571, WB 1:1000), FLK1 (Thermo Fisher Scientific MA5-15157, WB 1:1000), eNOS (Abcam AB76198, WB 1:1000), NLGN1 (Abcam ab153821, WB 1:1000), β-actin (R&D Systems MAB8928, WB 1:1000), and ZO-1 (Thermo Fisher Scientific 40-2200, ICC 1:200).

Techniques: Over Expression, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Analysis, Permeability, Tube Formation Assay, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting QKI-7 in vivo restores endothelial cell function in diabetes

doi: 10.1038/s41467-020-17468-y

Figure Lengend Snippet: Morphology of hiPSCs and their EC differentiated counterparts are shown by bright field microscopy. Scale bar: 50 μm ( a ). Flow cytometry showed the pure population of hiPS-derived ECs after MACS selection using CD144 magnetic beads ( b ). Immunofluorescence confocal image showing that the differentiated ECs expressed the EC-specific markers CD31, CD144, and ZO-1 localizing to cell–cell junction. QKI-7 displayed perinuclear cytoplasm localization. Scale bar: 25 μm ( c ). The expression of EC marker proteins CD31, CD144, KDR, and eNOS was shown by western blot ( d ). hiPS-ECs formed tube structure indicating their angiogenic capacity. Scale bar: 200 μm ( e ). Data are from n = 3 representative images. Source data are provided as a Source data file.

Article Snippet: Primary antibodies include QKI-7 (UC Davis/NIH NeuroMab Facility 73-200, WB 1:1000, ICC 1:100), CD144 (St John’s Laboratory STJ96234, WB 1:1000, ICC 1:200), CD31 (Abcam AB28364, WB 1:1000, ICC 1:20), KDR (R&D Systems MAB3571, WB 1:1000), FLK1 (Thermo Fisher Scientific MA5-15157, WB 1:1000), eNOS (Abcam AB76198, WB 1:1000), NLGN1 (Abcam ab153821, WB 1:1000), β-actin (R&D Systems MAB8928, WB 1:1000), and ZO-1 (Thermo Fisher Scientific 40-2200, ICC 1:200).

Techniques: Microscopy, Flow Cytometry, Derivative Assay, Selection, Magnetic Beads, Immunofluorescence, Expressing, Marker, Western Blot

Journal: Nature Communications

Article Title: Targeting QKI-7 in vivo restores endothelial cell function in diabetes

doi: 10.1038/s41467-020-17468-y

Figure Lengend Snippet: Compared with the non-diabetic iPS-EC HD19 control, iPS-EC P014 derived from a patient with diabetes showed significantly higher level of QKI-7 ( p value: 0.0083) accompanying decreased expression of CD144, NLGN1, and TSG6 ( p values: <0.0001) ( a ). When QKI-7 was knocked down ( p value: 0.0042), iPS-ECs showed increased expression of CD144, NLGN1, and TSG6 ( p values: 0.0006, 0.0081, 0.0019) ( b ). Comparison of protein level between diabetic iPS-EC P014 and non-diabetic HD19 was shown by western blot ( c ) and ELISA ( p value: 0.0005) ( d ). Upon QKI-7 knockdown, western blot showed increase of CD144 and NLGN1 ( e ) and ELISA verified the upregulation of TSG6 ( p value: 0.0011) ( f ). Diabetic iPS-EC P014 showed significantly lower RTCA cell index than non-diabetic HD19 indicating a permeability increase ( p value: 0.0009) ( g ). When QKI-7 was knocked down, P014 RTCA cell index was enhanced, showing the alleviation of cell barrier defect ( p value: 0.0019) ( h ). Compared with the non-diabetic HD19, the diabetic iPS-EC P014 displayed more THP-1 monocyte adhesion ( p value: 0.0203) ( i ) which was ameliorated by QKI-7 knockdown ( p value: 0.0163) ( j ). In tube formation assay, diabetic iPS-EC P014 formed less capillary structure than non-diabetic HD19 cells, measured by shorter tube branch length and smaller meshed area ( p values: 0.0312, 0.0052) ( k ) which was reversed by QKI-7 knockdown ( p values: 0.0061, 0.0225) ( l ), the data were normalized by setting the control group as 1. Scale bar i , j : 100 μm; scale bar k , l : 200 μm. Data are from three biologically independent experiments. Error bars represent mean ± SEM ( n = 3). P values are shown: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (two-tailed t test). Source data are provided as a Source data file.

Article Snippet: Primary antibodies include QKI-7 (UC Davis/NIH NeuroMab Facility 73-200, WB 1:1000, ICC 1:100), CD144 (St John’s Laboratory STJ96234, WB 1:1000, ICC 1:200), CD31 (Abcam AB28364, WB 1:1000, ICC 1:20), KDR (R&D Systems MAB3571, WB 1:1000), FLK1 (Thermo Fisher Scientific MA5-15157, WB 1:1000), eNOS (Abcam AB76198, WB 1:1000), NLGN1 (Abcam ab153821, WB 1:1000), β-actin (R&D Systems MAB8928, WB 1:1000), and ZO-1 (Thermo Fisher Scientific 40-2200, ICC 1:200).

Techniques: Control, Derivative Assay, Expressing, Comparison, Western Blot, Enzyme-linked Immunosorbent Assay, Knockdown, Permeability, Tube Formation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting QKI-7 in vivo restores endothelial cell function in diabetes

doi: 10.1038/s41467-020-17468-y

Figure Lengend Snippet: RNA immunoprecipitation (RIP) was carried out on P014 hiPS-ECs. Significantly more RNAs of CD144, TSG6, and NLGN1 were precipitated by QKI-7 antibody shown by real-time PCR and conventional PCR, indicating the direct binding of QKI-7 to transcripts of these three RNA targets ( p values: <0.0001) ( a – d ). When QKI-7 overexpressing hiPS-ECs or control cells (hiPS-ECs overexpressing an empty vector) were treated with actinomycin D in time point experiments from 0 to 12 h, the QKI-7 overexpression group showed lower mRNA level of CD144, TSG6, and NLGN1 at most of the time points as shown by the mRNA-decay curve, indicating that ectopic QKI-7 promoted mRNA degradation of the three targets ( e – g ). QKI-7 co-transfection with luciferase reporter plasmid containing wild-type 3′UTR of NLGN1 significantly suppressed the luciferase activity ( p value: 0.0329). While this effect was unobservable when QKI-7 was co-transfected with luciferase reporter plasmid containing mutated 3′UTR NLGN1 for the QKI motif ( h ). Data are from three biologically independent experiments. Error bars represent mean ± SEM ( n = 3). P values are shown: * p < 0.05, **** p < 0.0001, ns: not significant (two-tailed t test). Source data are provided as a Source data file.

Article Snippet: Primary antibodies include QKI-7 (UC Davis/NIH NeuroMab Facility 73-200, WB 1:1000, ICC 1:100), CD144 (St John’s Laboratory STJ96234, WB 1:1000, ICC 1:200), CD31 (Abcam AB28364, WB 1:1000, ICC 1:20), KDR (R&D Systems MAB3571, WB 1:1000), FLK1 (Thermo Fisher Scientific MA5-15157, WB 1:1000), eNOS (Abcam AB76198, WB 1:1000), NLGN1 (Abcam ab153821, WB 1:1000), β-actin (R&D Systems MAB8928, WB 1:1000), and ZO-1 (Thermo Fisher Scientific 40-2200, ICC 1:200).

Techniques: RNA Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Control, Plasmid Preparation, Over Expression, Cotransfection, Luciferase, Activity Assay, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Targeting QKI-7 in vivo restores endothelial cell function in diabetes

doi: 10.1038/s41467-020-17468-y

Figure Lengend Snippet: Matrigel plug assay was carried out using QKI-7 overexpressing miPS-ECs or control cells. In contrast to the control group, QKI-7 overexpression group formed much fewer capillary structures ( p value: 0.0096) with irregular organization, suggesting an interrupted angiogenetic capacity of ECs ( a images and quantification from 9 plugs per group based on n = 3 biological replicates). Hindlimb ischemia was induced in STZ-diabetic mice and QKI-7 overexpressing or control miPS-ECs were injected into the adductors immediately after induction of hind limb ischemia. Laser Doppler images of blood flow in the lower limbs of mice in prone position, with the ischemic leg highlighted by the yellow rectangle. After 14 days, delivery of control miPS-ECs significantly promoted the blood flow recovery of the ischemic hind limb, which was substantially compromised in the QKI-7 overexpression group ( p values: 0.0018, 0.0037) ( b ). In vivo QKI-7 knockdown was achieved by intramuscular injection of shRNA Lentivirus construct tagged with a CD144 promoter and GFP to target ECs, and blood flow recovery in STZ-diabetic mice was investigated. The QKI-7 knockdown group showed over 60% of perfusion ratio to the opposite intact limb which was significantly higher than the scrambled Lentivirus control group ( p value: 0.0081) ( c ). The effects of blood flow recovery in the QKI-7 knockdown group were also confirmed by hematoxylin and eosin staining where increased capillary density is shown ( p value: 0.0062) ( d ). The downregulation of QKI-7 by shRNA lentivirus was verified by immunohistochemistry, when the adductor tissue was harvested, fixed, cryosectioned, and stained. Compared with the scrambled control, the QKI-7 knockdown group showed significantly higher expression of EC marker CD144 along with QKI-7 suppression ( p values: 0.0072, 0.0056) ( e ). GFP staining clearly shown that the Lentiviral constructs tagged with GFP targeted ECs in vivo ( e ). Scale bar: a 200 μm; d 100 μm; e 200 μm. Data are from three biologically independent experiments. Error bars represent mean ± SEM ( n = 3). P values are shown: ** p < 0.01, ns: not significant (two-tailed t test). Source data are provided as a Source data file.

Article Snippet: Primary antibodies include QKI-7 (UC Davis/NIH NeuroMab Facility 73-200, WB 1:1000, ICC 1:100), CD144 (St John’s Laboratory STJ96234, WB 1:1000, ICC 1:200), CD31 (Abcam AB28364, WB 1:1000, ICC 1:20), KDR (R&D Systems MAB3571, WB 1:1000), FLK1 (Thermo Fisher Scientific MA5-15157, WB 1:1000), eNOS (Abcam AB76198, WB 1:1000), NLGN1 (Abcam ab153821, WB 1:1000), β-actin (R&D Systems MAB8928, WB 1:1000), and ZO-1 (Thermo Fisher Scientific 40-2200, ICC 1:200).

Techniques: Matrigel Assay, Control, Over Expression, Injection, In Vivo, Knockdown, shRNA, Construct, Staining, Immunohistochemistry, Expressing, Marker, Two Tailed Test

Journal: Critical Care

Article Title: Vasculotide reduces pulmonary hyperpermeability in experimental pneumococcal pneumonia

doi: 10.1186/s13054-017-1851-6

Figure Lengend Snippet: Vasculotide (VT) stabilized endothelial barrier function in vitro and ex vivo. a Confluent human pulmonary microvascular endothelial cells (hPMVEC) were preincubated for 90 minutes with 300 ng/ml VT or solvent (PBS) and stimulated with lipopolysaccharide (LPS; 0.1 μg/ml), pneumolysin (PLY; 0.25, 0.5, or 0.75 μg/ml), or PBS for 30 minutes. Cells were fixed and vascular endothelial (VE)-cadherin ( green ), actin fibers ( red ), and cell nuclei ( blue ) were stained for immunofluorescence microscopy. Unstimulated, PBS-treated, or VT-treated cells showed intact monolayers with tight intercellular contacts and thin actin fibers ( red ). In PBS-pretreated groups, the integrity between cell contacts was disrupted, and hPMVEC showed gap formation ( asterisks ) and stress fibers ( arrows ) after LPS or PLY stimulation ( upper row ). Pretreatment with VT stabilized the cell monolayer, avoided gap formation, and reduced stress fibers and cell loss from culture dishes ( lower row ). Representative images of three independent experiments are shown for every group. a Bar = 20 μm (valid for all photomicrographs). b Transcellular electrical resistance (TER) of isolated murine lung endothelial cell (mLEC) monolayers was continuously monitored. mLEC were pretreated for 30 minutes with VT (2, 10, or 50 ng/ml) or PBS and then stimulated with pneumolysin (PLY; 0.75 μg/ml). PLY stimulation decreased TER of mLEC monolayers, displaying loss of endothelial integrity ( solid curve ). Preincubation with VT attenuated the PLY-induced TER decrease in a dose-dependent manner ( dashed and dash-dotted curves ). c Ex vivo perfused and ventilated mouse lungs were pretreated with VT (50 ng/ml or 100 ng/ml) or PBS 15 minutes before PLY stimulation (1.4 μg/ml). After 30 minutes, lung vascular permeability was assessed by quantifying the concentration of continuously infused human serum albumin (HSA) in bronchoalveolar lavage fluid (BALF). Treatment with VT significantly decreased hyperpermeability of mouse lungs as compared with PBS treatment. Values are given as mean (C; n = 4–7) or mean + SEM (D; n = 6–8). ** p < 0.01, *** p < 0.001 between indicated groups

Article Snippet: The homogenate was filtered, collected by centrifugation (300 × g ), and washed twice, and the resulting cell suspension was incubated with antimouse VE-cadherin antibody (rat antimouse CD 144; BD Pharmingen, San Diego, CA, USA)-coated magnetic beads (sheep antirat IgG DynaBeads; Invitrogen GmbH, Karlsruhe, Germany) for positive isolation with the DynaMag-Spin magnet system (Invitrogen).

Techniques: In Vitro, Ex Vivo, Staining, Immunofluorescence, Microscopy, Isolation, Permeability, Concentration Assay

Journal:

Article Title: The lymphotoxin LT? 1 ? 2 controls postnatal and adult spleen marginal sinus vascular structure and function

doi: 10.1016/j.immuni.2009.01.010

Figure Lengend Snippet: Ten µm thick frozen sections from C57BL/6 spleens (n>10) were stained with anti-B220 (green) and either (A) anti-Flk-1 (red) or (B) anti-CD144 (VE-cadherin) (red) (magnification 200×). In panels C and D, staining was with anti-Flk-1 (green) and either (C) anti-PECAM-1 (red) or (D) anti-MOMA-1 (red). For panels E and F, spleen sections were cut at 20µm and stained with (E) anti-SMA alone (red) (magnification 100×) or together with (F) anti-B220 (green) (magnification 200×). For panels G and H, staining was with (G) anti-SMA alone (red) (magnification 100×) or together with (H) anti-Flk-1 (green) (magnification 630×). For panels C, D, G, and H, confocal microscopy was used to compile a series of Z-stack images to reconstruct a 6 µm thick section. CA, central arteriole; *, central arteriole branching vessel; (←), MS connecting vessels. Similar data were obtained in two additional experiments.

Article Snippet: The cells were incubated with anti-Flk-1 or anti-CD144 (BD Biosciences), followed by incubation with anti-rat Ig-MACS beads (Miltenyi Biotec) and positively selected on a magnetic MACS column.

Techniques: Staining, Confocal Microscopy

Journal:

Article Title: The lymphotoxin LT? 1 ? 2 controls postnatal and adult spleen marginal sinus vascular structure and function

doi: 10.1016/j.immuni.2009.01.010

Figure Lengend Snippet: Twenty µm thick frozen sections from spleens (n≥5) of the indicated mouse strains were stained with (A) anti-Thy1.2 (brown) and either anti-Flk-1 (blue, top panels) or anti-CD144 (VE-cadherin) (blue, bottom panels), and (B) anti-Thy1.2 (green) and anti-SMA (red) (magnification 200×). Inset images show magnified areas highlighting the Flk-1+ and CD144+ structures at the periphery of white pulp areas in the gene targeted mice.

Article Snippet: The cells were incubated with anti-Flk-1 or anti-CD144 (BD Biosciences), followed by incubation with anti-rat Ig-MACS beads (Miltenyi Biotec) and positively selected on a magnetic MACS column.

Techniques: Staining

Journal:

Article Title: The lymphotoxin LT? 1 ? 2 controls postnatal and adult spleen marginal sinus vascular structure and function

doi: 10.1016/j.immuni.2009.01.010

Figure Lengend Snippet: Spleens of Efnb2+/− (A, B) and Efnb2+/− Lta−/− (C, D) mice were cut to yield (A, C) 10 µm or (B, D) 120 µm sections and stained with anti-β-galactosidase (green) to visualize ephrinB2 expression via the expression of the in frame knock in of the LacZ gene. Confocal images were compiled to yield a 50–60 µm section of spleen, permitting analysis of the 3-D structure of a region of a single white pulp nodule (magnification 200×). Similar data were obtained by analyzing spleens from 5 additional pairs of mice. (E) The percentage of ephrinB2+ and Flk-1+ MS structures per WP area was quantified from 10 µm sections using a 20× objective. Graphs show mean + SD (n≥40 WP nodules using ≥5 spleens per strain). Data were compiled from 5 independent experiments.

Article Snippet: The cells were incubated with anti-Flk-1 or anti-CD144 (BD Biosciences), followed by incubation with anti-rat Ig-MACS beads (Miltenyi Biotec) and positively selected on a magnetic MACS column.

Techniques: Staining, Expressing, Knock-In

Journal:

Article Title: The lymphotoxin LT? 1 ? 2 controls postnatal and adult spleen marginal sinus vascular structure and function

doi: 10.1016/j.immuni.2009.01.010

Figure Lengend Snippet: (A) Frozen sections of spleens from C57BL/6 and Lta−/− mice harvested at the indicated ages (n≥5 per strain per time point) were stained with anti-Flk-1 (red), anti-SMA (green), and anti-B220 (blue) (magnification 200×). Expression of MAdCAM-1 protein was analyzed by (B) immunoblotting and (C) ELISA from extracts of whole spleens (n=10 spleens per strain per time point) of C57BL/6, Lta−/−, Ltbr−/− and Tnfrsf1a−/− mice. (D) Spleen sections from the indicated mouse strains (n=10 spleens per strain per time point) harvested on the indicated postnatal days were stained with anti-MAdCAM-1 (red) and anti-B220 (green). #p <0.01 comparing C57BL/6 and Tnfrsf1a−/− mice, *p <0.005 and **p <0.001 for comparisons between C57BL/6 and Ltbr−/− mice, and ϕp <0.001 for comparison between Tnfrsf1a−/− and Ltbr−/− mice.

Article Snippet: The cells were incubated with anti-Flk-1 or anti-CD144 (BD Biosciences), followed by incubation with anti-rat Ig-MACS beads (Miltenyi Biotec) and positively selected on a magnetic MACS column.

Techniques: Staining, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: The lymphotoxin LT? 1 ? 2 controls postnatal and adult spleen marginal sinus vascular structure and function

doi: 10.1016/j.immuni.2009.01.010

Figure Lengend Snippet: (A) Ten µm spleen sections from WT mice were stained with anti-LTβR (red) and anti-B220 (green) (n=3). Endothelial cells isolated from WT spleen (sECs) using either anti-Flk-1 or anti-CD144 were measured for (B) uptake of AcLDL (green) and (C) observed for formation of tube-like structures on Matrigel (n=3). sECs and the bEND.3 cell line were analyzed for expression of (D) LTβR protein and (E) RNA by immunoblotting and RT-PCR. (F) sECs isolated from WT and Lta−/− mice were plated on Matrigel containing a control GST antibody or an agonist anti-LTβR and cultured for 10 days. (G) bEND.3 cells were transfected with a control pBAP-FLAG vector (expressing bacterial alkaline phosphatase) or 0.1, 0.4, 1, and 4 µg of a pMAdCAM-1-FLAG vector. The cells were then plated on Matrigel and cultured for 24 hrs. (H) Protein extracts from transfected bEND.3 cells were prepared and an ELISA was performed to assess MAdCAM-1 protein expression. (I) Transfected bEND.3 cells grown on Anapore filters coated with Matrigel were fixed and frozen in O.C.T. and 8 µm cross-section slices were stained with anti-FLAG (red) and Hoechst (blue). Inset images show magnified areas highlighting the EC phenotype and amount of MAdCAM-1 associated with the Matrigel. Data shown are representative of 3 independent experiments.

Article Snippet: The cells were incubated with anti-Flk-1 or anti-CD144 (BD Biosciences), followed by incubation with anti-rat Ig-MACS beads (Miltenyi Biotec) and positively selected on a magnetic MACS column.

Techniques: Staining, Isolation, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: The lymphotoxin LT? 1 ? 2 controls postnatal and adult spleen marginal sinus vascular structure and function

doi: 10.1016/j.immuni.2009.01.010

Figure Lengend Snippet: (A) C57BL/6 adult mice were treated i.p. with either human IgG1 (huIg) or LTβR-Fc and spleens were harvested 2 wks later. 10 µm sections were stained with anti-Flk-1 (blue) and anti-B220 (brown). Similar results were obtained in 4 additional experiments. (B) The percentage of Flk-1+ MS structures per WP area was quantified from 10 µm sections using a 20× objective (n≥40 WP nodules from 3 independent experiments; graph shows mean + S.D.). (C) C57BL/6 mice were treated i.p. with either huIg or LTβR-Fc and 2 wks later were injected i.v. with 250 µg of TMR-labeled S. aureus bioparticles (red). Spleens were harvested at 3, 12 or 24 hrs after injection with the particles and sections were stained with anti-CD11b (green). Dashed lines outline WP areas. (D) The numbers of CD11b+ cells localized in the WP was quantified from ≥40 WP areas from 3 independent experiments with 3–5 mice per group. Graph shows mean + S.D. (E) Expression levels of the indicated cytokines, chemokines and adhesion molecules were analyzed by immunoblotting. Data are representative of at least 3 independent experiments with 3–5 mice per group.

Article Snippet: The cells were incubated with anti-Flk-1 or anti-CD144 (BD Biosciences), followed by incubation with anti-rat Ig-MACS beads (Miltenyi Biotec) and positively selected on a magnetic MACS column.

Techniques: Staining, Injection, Labeling, Expressing, Western Blot