Journal: iScience

Article Title: Macrophages treated with interferons induce different responses in lymphocytes via extracellular vesicles

doi: 10.1016/j.isci.2024.109960

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal anti CD11c (clone REAA618) Conjugated APC , Miltenyi , Cat#130-114-110.

Techniques: Recombinant, Protein Extraction, Protease Inhibitor, Pore Size, Electrophoresis, Staining, Software, Cytometry

Journal: Bioengineering & translational medicine

Article Title: 3D bioprinting of an implantable xeno-free vascularized human skin graft.

doi: 10.1002/btm2.10324

Figure Lengend Snippet: FIGURE 5 Phenotyping characterization of human keratinocytes under xeno-free conditions. (a) Live phasecontrast microscopy images of keratinocytes after isolation from the epidermis of donor foreskin and at the confluency state. (b) The cumulative population doublings of keratinocytes cultured under xeno-free conditions is comparable to KGM-Gold medium. (c) Flow cytometry analysis confirmed the expression of integrins α2β1, α5β1, α6, α3, and β4, but not αvβ3. (d) Confocal microscopy exhibiting CK14, CK10, junctional ZO-1, and intracellular occludin staining. Scale bar = 100 μm. Representative of three independent donors

Article Snippet: ECs, FBs, PCs, and KCs cultured under xeno-free conditions were analyzed for surface and intracellular markers expression by flow cytometry and immunofluorescence microscopy using antibodies against: CD31 (WM59; Biolegend), CD45 (2D1; Biolegend), ZO-1 (sc-33725; Santa Cruz), VE-cadherin (sc-6458; Santa Cruz), vWF (ab201336; abcam) claudin-5 (34-1600; Invitrogen), PDGFR-α (16A1; Biolegend), PDGFR-β (18A2; Biolegend), CD90 (5E10; Biolegend), NG2 (9.2.27; Invitrogen), a-SMA (1A4; Invitrogen), FAP (AF3715; Novus Biologics), integrins α2β1 (ab24697; abcam), α5β1 (NBP2-52680; Novus Biologics), αvβ3 (sc-7312; Santa Cruz), α6 (MAB13501; R&D systems), α3 (MAB1345; R&D systems), β4 (NBP1-43369), CK14 (LL002; Novus Biologics), CK10 (EP1607IHCY, abcam), and occludin.

Techniques: Microscopy, Isolation, Cell Culture, Flow Cytometry, Expressing, Confocal Microscopy, Staining

Journal: The Journal of Clinical Investigation

Article Title: Nonimmune cell–derived ICOS ligand functions as a renoprotective αvβ3 integrin–selective antagonist

doi: 10.1172/JCI123386

Figure Lengend Snippet: (A) Schematic of a gold surface with ICOSL protein on a sensor chip CM5 and associated protein (αvβ3 integrin) over which buffer is flown in SPR assay. (B–I) SPR sensorgrams depicting interaction of immobilized human ICOSL (hICOSL, B–D) or mouse ICOSL (mICOSL, E–I) with αvβ3 integrin. These bindings were tested in the presence (B and E, active form of αvβ3) or absence (C and F, inactive form of one with EDTA in the binding buffer) of Mn2+. (D and G) SPR used in an inhibition experiment with cRGDfv. Injection of αvβ3 integrin only (D, 120 nM αvβ3 or G, 150 nM αvβ3) resulted in a binding signal for immobilized hICOSL or mICOSL alone (pink line). Preincubation with cRGDfv (3 μM or 15 μM) significantly reduced the binding for ICOSL, indicating that the RGD peptide competes with ICOSL for binding to αvβ3 (orange line). cRGDfv alone was used as a control (green line). (H and I) SPR sensorgrams showing the binding between WT (H) or mutant (I) mICOSL protein and αvβ3 integrin in the presence of physiologically relevant divalent ions, Ca2+ (0.2 mM) and Mg2+ (0.1 mM). The average KD values were determined from at least 3 independent experiments. Rate constants (ka and kd) were determined by kinetic fitting (black dotted line) of the sensorgrams using 1-to-1 Langmuir binding equation, and KD values for B, E, and H were calculated by kd/ka (B, KD = 16.2 ± 4.0 nM for hICOSL/αvβ3 with Mn2+; E, KD = 24.2 ± 6.5 nM for mICOSL/αvβ3 with Mn2+; H, KD = 21.3 ± 1.2 nM for WT mICOSL/αvβ3 with Ca2+/Mg2+). KD values for C, F, and I were calculated from steady-state affinity fittings (C, KD = 411.8 ± 164.1 nM for hICOSL/αvβ3; F, KD ≥ 2 mM for mICOSL/αvβ3; I, KD = 0.83 ± 0.8 mM for mutant mICOSL/αvβ3 with Ca2+/Mg2+).

Article Snippet: Recombinant proteins used in this study were as follows: human integrin αvβ3 (R&D Systems, 3050-AV), human integrin α3β1 (R&D Systems, 2840-A3), human integrin αIIbβ3 (R&D Systems, 7148-A2), human integrin αvβ5 (R&D Systems, 2528-AV), human fibronectin (R&D Systems, 4305-FNB-200), human ICOSL-His Tag (Thermo Fisher Scientific, 11559H08H50), mouse integrin αvβ3 (R&D Systems, 7889-AV-050), mouse ICOSL (Sino Biological, 50-190-M08H25), human B7.1 (R&D Systems, 140-B1), and human ICOS (R&D Systems, 169-CS).

Techniques: SPR Assay, Binding Assay, Inhibition, Injection, Control, Mutagenesis

Journal: The Journal of Clinical Investigation

Article Title: Nonimmune cell–derived ICOS ligand functions as a renoprotective αvβ3 integrin–selective antagonist

doi: 10.1172/JCI123386

Figure Lengend Snippet: (A) Schematic representation of the protocol to measure relative cell adhesion levels in cultured human podocytes. Image analysis and quantification by high-content screening technology were described in Methods. (B) Phase-contrast microscopy images show that cultured human podocytes confer enhanced adhesion to ICOSL mediated by β3 integrin treated with Mn2+, but do not adhere on albumin (protein control). Increased adhesion levels were completely prevented by incubation with the integrin inhibitors, including cRGD peptide and anti-β3 integrin antibody. Scale bar 100 μm. (C) Quantification of the cell adhesion using the images in B. ICOSL induced cell adhesion to RGD-dependent β3 integrin on cultured podocytes. (D) Cell adhesion analysis of cultured podocytes plated on vitronectin. Data are shown as mean ± SD; ***P < 0.001; 1-way ANOVA with Tukey’s multiple comparison test (C and D).

Article Snippet: Recombinant proteins used in this study were as follows: human integrin αvβ3 (R&D Systems, 3050-AV), human integrin α3β1 (R&D Systems, 2840-A3), human integrin αIIbβ3 (R&D Systems, 7148-A2), human integrin αvβ5 (R&D Systems, 2528-AV), human fibronectin (R&D Systems, 4305-FNB-200), human ICOSL-His Tag (Thermo Fisher Scientific, 11559H08H50), mouse integrin αvβ3 (R&D Systems, 7889-AV-050), mouse ICOSL (Sino Biological, 50-190-M08H25), human B7.1 (R&D Systems, 140-B1), and human ICOS (R&D Systems, 169-CS).

Techniques: Cell Culture, High Content Screening, Microscopy, Control, Incubation, Comparison

Journal: The Journal of Clinical Investigation

Article Title: Nonimmune cell–derived ICOS ligand functions as a renoprotective αvβ3 integrin–selective antagonist

doi: 10.1172/JCI123386

Figure Lengend Snippet: In this study, it is shown that ICOSL binds podocyte αvβ3 integrin through its RGD motif. Kidney injury results in a rapid increase of ICOSL expression, leading to podocyte protection by blocking active αvβ3 integrin. ICOSL acts as a regulatory brake to modulate active αvβ3 integrin–mediated signaling. Fp, foot process.

Article Snippet: Recombinant proteins used in this study were as follows: human integrin αvβ3 (R&D Systems, 3050-AV), human integrin α3β1 (R&D Systems, 2840-A3), human integrin αIIbβ3 (R&D Systems, 7148-A2), human integrin αvβ5 (R&D Systems, 2528-AV), human fibronectin (R&D Systems, 4305-FNB-200), human ICOSL-His Tag (Thermo Fisher Scientific, 11559H08H50), mouse integrin αvβ3 (R&D Systems, 7889-AV-050), mouse ICOSL (Sino Biological, 50-190-M08H25), human B7.1 (R&D Systems, 140-B1), and human ICOS (R&D Systems, 169-CS).

Techniques: Expressing, Blocking Assay

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 1 Vps26a deficiency leads to the maintenance of stemness during ESC-mediated neurogenesis. a Effect of Vps26a deficiency on changes in alkaline phosphatase (AP) staining during neural differentiation (ND). Wild-type (WT; +/+) and Vps26a-/- (-/-) ESCs were differ- entiated in neurobasal medium (NBM) for the indicated time periods and subjected to AP staining. Cell clusters with differentiated morphologies are indicated by yellow dotted lines. Scale bar, 50 μm. b AP activities of WT and Vps26a-/- ESCs differentiated for 6 days indicated by scoring of the signal intensities of at least 60 colonies from three independent experiments. c The number of WT and Vps26a-/- cells during ND for 8 days. Error bars indicate the means ± standard deviation (SD; n = 3). ***P < 0.001 compared with WT cells each day during ND. d, e Effect of Vps26a deficiency on expression

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Staining, Standard Deviation, Expressing

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 2 Vps26a knockdown prolongs the maintenance of stemness under ND conditions in P19 ECCs. a, b The effect of Vps26a knockdown on the expression of ESC stemness genes were determined by semi-qPCR (a) and western blot (b) analyses of Oct3/4 and Nanog using shCTL- and shVps26a-ECCs. c Morphological changes of shCTL (shC)- and shVps26a (shV)-ECCs during retinoic acid-induced neurogenesis (RA-ND) for the indicated time periods. The yellow arrowheads indicate cells with differentiated morphologies. d–f Expression kinetics of ESC stemness and neuronal markers examined by semi-qPCR (d), qPCR (e), and western blotting (f)

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Knockdown, Expressing, Western Blot

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 4 Vps26a is required for increased ROS leading to ESC-mediated neurogenesis. a, b The effect of Vps26a deficiency (a) or knockdown (b) on ROS generation was determined by flow cytometry using WT and Vps26a-/- ESCs (a) and shCTL- and shVps26a-ECCs (#1, #10, and #14) (b) differentiated for 6 days and 48 h, respectively. Unstained cells (black lines) were used as a negative control. The data are representative of at least three independent experiments and presented as means ± SD (n = 3). ***P < 0.001. c WT and Vps26a-/- ESCs were differentiated in the presence or absence of 2.5 mM NAC for 6 days, and ROS levels were measured by flow cytometry. The data are

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Knockdown, Cytometry, Negative Control

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 5 Vps26a involves Nox expression during neurogenesis. a, b The effect of Vps26a deficiency and knockdown on the expression of the Nox family was determined by semi-qPCR (a) and qPCR (b) analyses using shCTL (shC)- and shVps26a (shV)-ECCs, differentiated for the indicated time periods. Error bars indicate the means ± SD (n = 3). **P < 0.01; ***P < 0.001 compared with shCTL-ECCs each day. c Over- view of Vps26a and Nox4 immunostaining of a sagittal section of WT

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Expressing, Knockdown, Immunostaining

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 6 Identification of the interaction between Vps26a and Nox4. a Serial sagittal sections of WT embryos were immunostained for Vps26a, Nox4, and Tubb3 and counterstained lightly with hematox- ylin at E10.5. da, dorsal aorta; l, lung; h, heart; nc, notochord; nl, neural lumen; nt, neural tube; som, somite. b Vps26a immunopreci- pitation followed by anti-Vps26a and -Nox4 immunoblots using lysates obtained from WT (+/+) and Vps26a-/- (-/-) ESCs during neural differentiation for 0 or 6 days. IgG was used as an immuno- precipitation control. c GST-tagged Vps26a was subjected to a pull- down assay with the lysates of HEK293 cells transfected with HA- Nox4C (C-terminal region, 249–574 aa)-expressing plasmid. Immu- noblot analysis with anti-HA antibody is shown at the top. Equal

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Western Blot, Immunoprecipitation, Control, Pull Down Assay, Transfection, Expressing, Plasmid Preparation

Journal: Cell death and differentiation

Article Title: Novel crosstalk between Vps26a and Nox4 signaling during neurogenesis.

doi: 10.1038/s41418-018-0226-0

Figure Lengend Snippet: Fig. 7 Nox4-generated ROS lead to a loss of stemness and subsequent neurogenesis from ESCs. a, b The effects of antioxidant and Nox inhibitor treatments on ROS generation (a) and ESC stemness tran- scription levels (b) were examined by flow cytometry using WT and Vps26a-/- ESCs differentiated in the presence or absence of 2.5 mM NAC or 10 μM DPI for 6 days. Error bars indicate the means ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001 compared with no treat- ment. c Double-label immunocytochemical analysis of Vps26a (red),

Article Snippet: For construction of the C-terminally GST-tagged Vps26a, the cDNA encoding GST and Vps26a WT (1–360 aa), or deletion constructs containing amino acids 1–148, 149–245, or 246–360, were amplified and inserted into the pEBG-GST mammalian expression vector (Addgene #22227).

Techniques: Generated, Cytometry