Journal: bioRxiv

Article Title: High-throughput differentiation of human blood vessel organoids reveals overlapping and distinct functions of the cerebral cavernous malformation proteins

doi: 10.1101/2024.12.04.626588

Figure Lengend Snippet: High-throughput (HT)-compatible and nearly xeno-free synthesis of vascular networks and blood vessel organoids from fluorescently tagged human induced pluripotent stem cells (hiPSCs). A Schematic illustration of the new differentiation protocol and representative images for the main differentiation steps (scale bars: d-2, d0, d3, d5 = 100 µm; d8, d12 = 250 µm; d14, d17 = 500µm). B Shown are the steps of embedding the vascular aggregates in an Akura 96-well plate and transferring the vascular networks from the Akura 96-well plate to a PrimeSurface 96 Slit-well plate. C The use of PrimeSurface 96 Slit-well plates reduces the time required for medium exchange (left image). Akura 96-well plates allow aggregates to be embedded in small cavities, minimizing the matrix surrounding the vascular networks (black arrows) and allowing direct transfer of vascular networks (white arrows) to new plates without time-consuming manual extraction of the networks from the gel (middle and right images). D The new protocol is simple to handle and achieves high synthesis efficiency after minimal training. Shown are the efficiencies of three training runs. E The sprouting efficiency is maintained when fetal bovine serum (FBS) is replaced with human platelet lysate (hPL) or chemically defined Panexin CD (PCD). The total numbers of sufficiently sprouted networks and vascular aggregates with insufficient sprouting are written inside the bars. F,G HiPSC-derived vascular networks (F) and blood vessel organoids (G) differentiated with the HT-compatible and nearly xeno-free protocol consist of a complex network of endothelial cells (CD31) and associated pericytes (PDGFR-β) [representative images; scale bars: 50 µm (F); 200 µm (G)]. White arrowheads indicate angiogenic sprouts. G Perfusion of vascular networks with TMR-amino-dextran in OrganoPlate graft plates shows anastomoses between the GFP-labeled vascular networks and the HUVEC-derived vascular bed (top, white arrowheads) as well as correct formation and permeability of the vascular networks (bottom).

Article Snippet: Human umbilical vein endothelial cells (HUVECs, PromoCell, Heidelberg, Germany) were cultured in endothelial cell growth medium (ECGM, PromoCell) supplemented with 10 % fetal bovine serum (FBS) (Thermo Fisher Scientific, #A5670701).

Techniques: High Throughput Screening Assay, Transferring, Extraction, Derivative Assay, Labeling, Permeability

Journal: bioRxiv

Article Title: High-throughput differentiation of human blood vessel organoids reveals overlapping and distinct functions of the cerebral cavernous malformation proteins

doi: 10.1101/2024.12.04.626588

Figure Lengend Snippet: Changes in cellular composition of KO blood vessel organoids. A Experimental design of the single cell RNA sequencing (scRNA-seq) experiments for KO and WT organoids. B Uniform Manifold Approximation and Projection (UMAP) visualization of the scRNA-seq data with cells colored according to the unsupervised clustering results. C UMAP plots colored by the expression levels of marker genes predominantly expressed in endothelial, pericyte, smooth muscle and proliferating cells. Purple color indicates high expression. Low expression is indicated by grey color. D Separate UMAP plots of scRNA-seq data from CCM1 KO, CCM2 KO, CCM3 KO, and WT blood vessel organoids. The numbers represent the different clusters. E The composition of the clusters is illustrated by the proportion of CCM1 KO, CCM2 KO, CCM3 KO, and WT cells within each cluster. F The cellular composition of KO and WT blood vessel organoids is illustrated by the distribution of CCM1 KO, CCM2 KO, CCM3 KO, and WT samples into the different clusters.

Article Snippet: Human umbilical vein endothelial cells (HUVECs, PromoCell, Heidelberg, Germany) were cultured in endothelial cell growth medium (ECGM, PromoCell) supplemented with 10 % fetal bovine serum (FBS) (Thermo Fisher Scientific, #A5670701).

Techniques: RNA Sequencing Assay, Expressing, Marker

Journal: Cell

Article Title: Cannabinoid receptor 1 antagonist genistein attenuates marijuana-induced vascular inflammation

doi: 10.1016/j.cell.2022.04.005

Figure Lengend Snippet: (A) The effects of Δ9-THC on cell viability of human coronary artery endothelial cells (HCAECs), human umbilical vein endothelial cells (HUVECs), normal human cardiac fibroblasts-ventricular (NHCF-V), and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Cells were treated with increasing concentrations of Δ9-THC for 48 h, and cell viability was measured by the CellTiter-Glo luminescent cell viability assay.

Article Snippet: Cell culture We obtained the following: human umbilical vein endothelial cells (HUVECs), human coronary artery endothelial cells (HCAECs), normal human cardiac fibroblasts-ventricular (NHCF-V) cells, human erythroleukemia (HEL 92.1.7), and human neuroblastoma cells (SK-N-FI) from the American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Cell Viability Assay

Journal: Frontiers in Immunology

Article Title: RA Fibroblast-Like Synoviocytes Derived Extracellular Vesicles Promote Angiogenesis by miRNA-1972 Targeting p53/mTOR Signaling in Vascular Endotheliocyte

doi: 10.3389/fimmu.2022.793855

Figure Lengend Snippet: Identification and intracellular localization of RA FLSs derived EVs. RA FLSs were obtained from RA patients. Cells were maintained in DMEM and passages 3-6 were used for the follow-up experiments. EVs were prepared as the established procedure. (A) Representative image of EVs photographed using transmission electron microscope (TEM). (B) Representative results of nanoparticle tracking analyses of EVs. (C) EVs markers of CD9, CD63, CD81 and TSG101 were showed by Western Blot. (D) Immunofluorescence image to show the interaction between ECs and EVs (Nucleus: blue, EVs: green). FLSs, fibroblast-like synoviocytes; ECs, endothelial cells; EVs, extracellular vesicles.

Article Snippet: Human umbilical vein endothelial cell line (EA. hy926) , was purchased from ATCC.

Techniques: Derivative Assay, Transmission Assay, Microscopy, Western Blot, Immunofluorescence

Journal: Frontiers in Immunology

Article Title: RA Fibroblast-Like Synoviocytes Derived Extracellular Vesicles Promote Angiogenesis by miRNA-1972 Targeting p53/mTOR Signaling in Vascular Endotheliocyte

doi: 10.3389/fimmu.2022.793855

Figure Lengend Snippet: RA FLSs derived EVs promote tube formation in ECs in vitro and in vivo . (A–C) RA FLSs or Trauma FLSs derived EVs were obtained using the established procedure and cultured with ECs in tubule formation system or migration system. For LPS stimulation, RA FLSs were cultured in DMEM with LPS (1 μg/mL) for 3 days. (A) The tubule formation capacity of RA FLSs derived EVs was determined. Representative photos (50×) were obtained using microscope imaging (panel upper). The results were summarized by calculating the related parameters (panel lower). (B) The dose-dependent effect of RA FLSs derived EVs stimulation of ECs tubule formation in vitro . The relative parameters are given. The line graph describes a summary of experiments at relative parameters as indicated (n = 3). (C) The migration capacity of RA FLSs derived EVs was determined by transwell chamber system. The final concentration of EVs was 50 μg/mL. Typical photos (100×) (panel left), summary data (panel right) are shown. The data indicate the mean ± S.E.M of three independent experiments. (D, E) FLSs derived EVs expedite tubule genesis in matrigel angiogenesis mice model. Matrigel mixed with ECs and EVs was injected subcutaneously into NOD SCID mice. Two weeks post the transplantation, blood vessels were analyzed with Masson and CD31 fluorescence staining. (D) Masson staining results. Typical photos (100×) (panel upper), summary data (panel lower) are shown. (Red represented endothelial cells and blood vessels, blue represented matrix gel; n=3). (E) CD31 fluorescence staining. Typical photos (100×) are shown. FLSs, fibroblast-like synoviocytes; ECs, endothelial cells; EVs, extracellular vesicles; LPS, lipopolysaccharide. (*P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: Human umbilical vein endothelial cell line (EA. hy926) , was purchased from ATCC.

Techniques: Derivative Assay, In Vitro, In Vivo, Cell Culture, Migration, Microscopy, Imaging, Concentration Assay, Injection, Transplantation Assay, Fluorescence, Staining

Journal: Frontiers in Immunology

Article Title: RA Fibroblast-Like Synoviocytes Derived Extracellular Vesicles Promote Angiogenesis by miRNA-1972 Targeting p53/mTOR Signaling in Vascular Endotheliocyte

doi: 10.3389/fimmu.2022.793855

Figure Lengend Snippet: RNAs parts in RA FLSs derived EVs played a pivotal role in promoting ECs angiogenesis. RA FLSs were stimulated with LPS for 3 days. EVs were then obtained using the established procedure. For RNA or protein degradation, EVs were incubated with RNase A or proteinase K. The angiogenesis capacities of EVs were evaluated using the established in vitro systems and mice model. (A) The tubule formation capacity of RA FLSs derived EVs was determined. Representative photos (50×) were obtained using microscope imaging (panel upper). The results were summarized by calculating the related parameters (panel lower). (B) The migration capacity of RA FLSs derived EVs was determined by transwell chamber system. The final concentration of EVs was 50 μg/mL. Typical photos (100×) (panel left), summary data (panel right) are shown. The data indicate the mean ± S.E.M of three independent experiments. (C, D) Access of EVs tubule genesis in mice model. (C) Masson staining results. Typical photos (100×) (panel upper), summary data (panel lower) are shown. (Red represented endothelial cells and blood vessels, blue represented matrix gel; n=3). (D) CD31 fluorescence staining. Typical photos (100×) are shown. (*P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant).

Article Snippet: Human umbilical vein endothelial cell line (EA. hy926) , was purchased from ATCC.

Techniques: Derivative Assay, Incubation, In Vitro, Microscopy, Imaging, Migration, Concentration Assay, Staining, Fluorescence

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: PSMA was detected in HUVECs cultured with the conditioned medium derived from LNCaP cells. (A) Confocal images of prostate cancer cells (PC3, DU145, LNCaP cells) cultured in condition 1 or condition 2. The scheme of each condition by which each conditioned medium (CM) was prepared is shown on the right. To prepare the CM, 1.5 × 10 5 cells of prostate cancer cells were seeded on 6‐well plastic dishes (condition 1) or collagen I gels (condition 2). Three days later, the media were replaced with fresh media. The cells were then incubated for another 3 days, then the media were collected as CM. Bars: 100 µm. (B, C) Confocal images of HUVECs cultured with the CM derived from LNCaP cells (B) or PC3 and DU145 cells (C). The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. Bars: 100 µm. (D) Western blots of HUVEC lysates cultured with the CM derived from prostate cancer cells in condition 2 for 72 h. The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. The lysates from LNCaP cells were used as a positive control of PSMA expression. (E) The mRNA expression of PSMA in HUVECs cultured with the CM derived from LNCaP cells in condition 2 for 72 h. The CM was diluted to half of its concentration with EBM‐2. The resulting solution was added to the HUVECs seeded on the collagen I gels. Data are mean ± SEM from three independent experiments. ** p < .01. HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Cell Culture, Derivative Assay, Incubation, Concentration Assay, Immunofluorescence, Staining, Western Blot, Positive Control, Expressing

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: Fractionation of the CM derived from LNCaP cells. (A) Confocal images of HUVECs cultured with each fraction of CM derived from LNCaP cells. Each fraction was diluted to half of its concentration with EBM‐2 or suspended with EBM‐2, and added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. The PSMA‐positive HUVECs were shown by arrowheads. Bars: 100 µm. The representative images from three independent experiments were shown. (B) Confocal images of HUVECs cultured with 10,000 g pellet fractions of CM derived from LNCaP cells. The 10,000 g pellet fraction was labeled with a membrane marker dye, before the suspension in EBM‐2 medium. The labeled 10,000 g pellet was cultured with HUVECs for 6 h, and cells were subjected to immunofluorescence staining for PSMA. Bars: 10 µm (left) and 2 µm (right; magnified images of squares in left images). The representative images from three independent experiments were shown. (C) Western blots of 10,000 g pellet fraction of CM derived from LNCaP cells. The lysates from HUVECs and LNCaP cells were used as negative and positive controls of PSMA expression, respectively. The representative blot data from three independent experiments were shown. (D) Confocal images of HUVECs cultured with the CM derived from PC3 cells that stably express Myc‐PSMA or PSMA‐myc. The CM was diluted in half with EBM‐2, and added to the HUVECs seeded on the collagen I gels. Seventy‐two hours later, cells were subjected to immunofluorescence staining for PSMA. Bars: 100 µm. The representative images from three independent experiments were shown. CM, conditioned medium; HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Fractionation, Derivative Assay, Cell Culture, Concentration Assay, Immunofluorescence, Staining, Labeling, Marker, Western Blot, Expressing, Stable Transfection

Journal: The Prostate

Article Title: PSMA‐positive membranes secreted from prostate cancer cells have potency to transform vascular endothelial cells into an angiogenic state

doi: 10.1002/pros.24237

Figure Lengend Snippet: The tube formation assay of HUVECs cultured with 10,000 g pellet fraction of CM derived from prostate cancer cells. (A) Representative images of tube formation. HUVECs seeded on collagen I gel were treated with the 10,000 g pellet fraction of CM derived from LNCaP cells for 6 h, and packed on collagen I followed by VEGF‐A stimulation for 66 h. HUVECs were stained with Calcein‐AM before acquisition of images. To examine the PSMA dependency, the CM was prepared from LNCaP cells depleted of PSMA. Bars: 100 µm. (B) The quantitation of (A). Total tube lengths from three independent experiments were measured and normalized to those of cells cultured with normal EBM‐2. Data are the means ± SEM . * p < .05; n.s., not significant. (C) Western blots of LNCaP cell lysates, 72 h posttransfection with the indicated siRNAs. (D) Confocal images of PC3 cells stably expressing Myc‐PSMA or PSMA‐myc. Bars: 100 µm. (E) Representative images of tube formation. HUVECs seeded on collagen I gel were treated with the 10,000 g pellet fraction of CM derived from PC3 cells for 6 h, and packed in collagen I followed by VEGF‐A stimulation for 66 h. HUVECs were stained with Calcein‐AM before acquisition of images. Bars: 100 µm. (F) The quantitation of (E). Total tube lengths from three independent experiments were measured and normalized to those of PC3 (parental). Data are the means ± SEM . * p < .05; ** p < .01. CM, conditioned medium; HUVEC, human umbilical vascular endothelial cell; mRNA, messenger RNA; PSMA, prostate‐specific membrane antigen [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Human umbilical vascular endothelial cells (HUVECs) were purchased from Lonza.

Techniques: Tube Formation Assay, Cell Culture, Derivative Assay, Staining, Quantitation Assay, Western Blot, Stable Transfection, Expressing

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Schematic of flow sorting of CD31+CD45− BECs from mouse cortex and hippocampi. n=6 young and 6 aged biologically independent samples; each sample= 2 biologically independent mice cortex/hippocampi pooled as one sample. There were 1006 significant differentially expressed genes (*q<0.05, Cuffdiff Statistical Package61). (b) FACS gating strategy to isolate single BECs. PI+ dead cells were excluded. CD11b+ and CD45+ cells were gated to exclude monocytes/macrophages and microglia. CD31+Cd11b−CD45− cells were defined as the BEC population. (c) Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of CNS cell-type specific markers. n=6 young and 6 aged biologically independent samples. Mean +/− SEM. (d) FPKM values of leukocyte binding adhesion molecules including Vcam1. n=6 young and 6 aged biologically independent samples. Bars represent mean. Error bars derived from SEM. Specific q values shown are derived from Cuffdiff Statistical Package (*q=0.0015). See and Source Data for details. (e) FPKM values of tight junction genes. n=6 young and 6 aged biologically independent samples. Mean +/− SEM. q=0.16, *q=0.0013, **q=0.0015, Cuffdiff Statistical Package. See and Source Data for details. (f) FPKM values of the gene transcripts in murine young and aged CD31+BECs of human plasma proteins that change with age (see for list of human plasma proteins expressed in murine BECs). n=6 young and 6 aged biologically independent samples. Mean +/− SEM. *q=0.0015, **q=0.021, Cuffdiff Statistical Package. See and Source Data for details. (g) C57BL6 mice were injected with anti-VCAM1-DL488 or IgG-DL488 isotype control (r.o.) 2 hours before perfusion to label BECs in vivo prior to brain dissociation, staining and FACS. (h) Flow gating and histogram plots of pooled (n=4 mice/ age group), young or aged hippocampi isolated from healthy mice injected with fluorescently tagged DL488 anti-VCAM1 mAb or IgG-DL488 conjugated isotype control as depicted in (g). (i) Quantification of CD31+VCAM1+cells isolated from (left) healthy cortex (n=4 mice per age group, individually measured) and (right) 4 technical replicates of hippocampi that are pooled from 4 mice per age group. Mean +/− SEM. *p=0.0015. Two-tailed Student’s t-test. (j) sVCAM1 ELISA in plasma from young isochronic or heterochronic parabionts following 5 weeks of parabiosis. n=11 mice/group pooled from two independent experiments. **p=0.0031,Two-tailed Student’s t-test. Mean +/− SEM. (k) Confocal images in the DG of VCAM1, lectin, and Aqp4 of young isochronic or heterochronic parabionts 5 weeks after surgery. Quantification shown in . Hoechst labels cell nuclei. Scale bar = 100 μm. n= 8 mice in the Young isochronic group and 13 mice in Young heterochronic group from two independent experiments; representative images are shown. (l) Boxplot of expression levels of classical pan-endothelial and BBB-specific transcripts (n=272 BECs total). Minima, maxima, median, and percentiles are listed in . (n=146 Capillary BECs, n=59 Venous BECs, n=67 Arterial BECs pooled from 8 mice hippocampi). (m) Overlay of Vcam1 mRNA levels on corresponding coordinate on the Cd31 vs Vcam1 fluorescent intensity plots obtained during FACs sorting. (n) Validation of the correlation (Spearman’s rho = 0.704) between protein and mRNA levels of 77 single BECs sorted from both Vcam1+ and Vcam1− gates. Scatterplot of Vcam1 fluorescence intensity as measured by FACs and corresponding transcript counts (per million). (o) tSNE visualization colored by cell identity (aged vs. young) (n=160 young BECs, n=112 aged BECs pooled from 4 mice hippocampi per age group). (p) Comparison of Vcam1 expression levels in young and aged hippocampal CD31+ BECs collected from the VCAM1+ gate during FACs sorting (bars represent mean and error bars = SD). (n=160 young BECs, n=112 aged BECs pooled from 4 mice hippocampi per age group). *p=0.017. Two-tailed Mann-Whitney test. (q) Violin plots of mRNA expression levels of Icam1 in all isolated BECs (bottom) and specifically in VCAM1+ enriched BECs (top). Other adhesion molecules, namely Psele and Sele were not found to be expressed in isolated CD31+ BECs. (All BECs: n=160 young BECs, n=112 aged BECs pooled from 4 mice hippocampi per age group; VCAM1+ enriched BECs: n=56 Vcam1+ young BECs, n=44 Vcam1+ Aged BECs pooled from 4 mice hippocampi per age group). Minima, maxima, median, and percentiles are listed in . (r) Violin plots of tight junction markers in all isolated young and aged BECs. (n=160 young BECs, n=112 aged BECs pooled from 4 mice hippocampi per age group). Minima, maxima, median, and percentiles are listed in .

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Binding Assay, Derivative Assay, Clinical Proteomics, Injection, Control, In Vivo, Staining, Isolation, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Expressing, Biomarker Discovery, Fluorescence, Comparison, MANN-WHITNEY

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Heat map displaying up or down-regulated genes in young versus aged BECs based on bulk RNAseq (n=6 young and 6 aged biologically independent samples; each sample= 2 biologically independent mice cortex/hippocampi pooled as one sample). There were 1006 significant differentially expressed genes (*q<0.05, Cuffdiff Statistical Package ). (b) Fragments Per Kilobase of transcript per Million mapped reads (FPKM) of BEC cell-type specific markers. n=6 young and 6 aged biologically independent samples. Mean +/− SEM. (c) FPKM values of inflammation and activation related genes. n=6 young and 6 aged biologically independent samples. Mean +/− SEM. Specific q values shown are derived from Cuffdiff Statistical Package. See and Source Data for details. (d) Heat map showing changes in 31 out of 74 human plasma factors with aging (p<0.05, Spearman’s correlation coefficient). Multiplex assay used (n=118 healthy humans). (e) Spearman correlation of VCAM1 levels and age (Spearman’s correlation coefficient = 0.47; p=7.7e-08; q=5.72 × 10 −6 ). (f) Human sVCAM1 ELISAs in 11 young (<25 years old) or 11 aged (>65 years old) plasma from individual healthy donors. **p=0.0033, Student’s t-test . Two-tailed. Mean +/− SEM. (g) ELISA for mouse sVCAM1 in plasma from young (3-month-old; n=8), middle-aged (8–10-month-old; n=10), and aged (19-month-old; n=8) mice. Mean +/− SEM. ***p=0.0001 ****p<0.0001, 1-way ANOVA with Tukey’s multiple comparisons test. (h) Representative confocal images in the DG of young (3-month-old) or aged (18-month-old) mice given retro-orbital (r.o.) injections of fluorescently conjugated anti-VCAM1 and anti-Meca99 2 hours before perfusion. Hoechst labels cell nuclei. Scale bar = 50 µm. 3D rendering of the 2D images are displayed. 3D Scale bar = 50 µm. VCAM1 quantified in 4 separate cohorts of mice spaced 6 months or more apart. (i) Quantification of VCAM1+Lectin+ stained brain vasculature in young, middle, and aged hippocampi. n=12 young (3-4-month-old), 5 middle (12-month-old), 11 aged (18-month-old), and 6 very aged (24-month-old) mice. VCAM1 quantified in 4 separate cohorts of mice spaced 6 months or more apart. Mean +/− SEM. ***p=0.0002, ****p<0.0001, 1-way ANOVA with Tukey’s multiple comparisons test. (j) Quantification in the DG of VCAM1+Lectin+ stained brain vasculature of young isochronic or heterochronic parabionts 5 weeks after surgery. Representative images shown in . **p=0.0071, Student’s t-test . Two-tailed. Mean +/− SEM. n= 8 mice in the Young Isochronic group and 13 mice in Young heterochronic group from two independent experiments.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Activation Assay, Derivative Assay, Clinical Proteomics, Multiplex Assay, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Staining

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Schematic of the Blood-brain barrier (BBB). Nutrient-rich, oxygenated blood is pumped into the brain through cerebral arterial BECs (arteries and arterioles), which are protected and supported by smooth muscle cells (SMCs) that cover the endothelium and form a basement membrane layered by astrocytic end-feet of the brain parenchyma. The blood is transferred to highly specialized capillaries, which are comprised of BECs that form unique tight junctions and are wrapped by pericytes (Peric.) within the endothelial basement membrane, which is then covered by astrocytic end-feet. BBB capillaries are the site of controlled transport of fluids and solutes into the CNS. Immuno-surveillance and occasional extravasation of leukocytes (Leuk.) into the CNS parenchyma occurs at the level of postcapillary venous cells (venules and veins) the vascular segments into which blood flows after passing through the capillaries. Postcapillary Venules contain enlarged perivascular space between the endothelial and astrocytic basement membranes where occasional immune cells can reside. , (b) Unbiased clustering of 112 aged and 160 young hippocampal BECs using whole transcriptome and visualization with tSNE reveals 3 molecularly distinct BEC populations. (c) Violin plots of Vcam1 reveal differing levels of the transcript in each of the cell clusters. Minima, maxima, median, and percentiles are listed in . (n=146 Capillary BECs, n=59 Venous BECs, n=67 Arterial BECs pooled from 8 mice hippocampi). (d) Dotplot comparing the expression (scaled transcript counts and percent of population expressing) of various classical inflammatory, Notch signaling, arteriolar, venular and capillary markers between the three clusters (Cluster 0: Vcam1 -negative, Cluster 1: Vcam1 -pos, Cluster 2: Vcam1 -pos). (e) Heatmap of the scaled expression of the top 10 enriched genes (differentially expressed with p<0.05, Mann-Whitney test, two-sided) in each cluster. Genes are ranked by highest log-fold change when compared to all other cells. (f) tSNE visualization colored by Vcam1 expression levels. Clusters are further annotated by their putative functional-phenotype and vessel segmental identity. (n=146 Capillary BECs, n=59 Venous BECs, n=67 Arterial BECs pooled from 8 mice hippocampi). (g) GeneAnalytics (GSEA Package)- Brain Endothelial Cell Pathway analysis of the Vcam1−positive venous and arteriolar hippocampal BEC clusters. The top 10 pathways containing Vcam1 are highlighted here, along with the number of genes in each pathway enriched and the score assigned to each pathway. (h) Violin plots of various inflammation-related genes in each of the 3 distinct clusters. To note, age-related chemokine Ccl11 and its receptor, Ccr3, were not found to be expressed in isolated CD31+ BECs. Minima, maxima, median, and percentiles are listed in . (n=146 Capillary BECs, n=59 Venous BECs, n=67 Arterial BECs pooled from 8 mice hippocampi). (i) Violin plots of cytokine receptors enriched in the Vcam1−positive venous cluster. Minima, maxima, median, and percentiles are listed in . (n=146 Capillary BECs, n=59 Venous BECs, n=67 Arterial BECs pooled from 8 mice hippocampi). (j) Young (2.5-month-old) mice were injected with PBS control (n=5 mice high dose, 3 mice low dose), TNF-α (n=3 mice at high dose, 4 mice at low dose), IL-1β (n=4 mice low dose, 4 mice high dose), or IL-6 r.o. (n=4 mice low dose) daily over 5 days (2 µg per injection; low dose) or acutely (10 µg; high dose). Representative confocal images (bottom) and quantification (top) of VCAM1+ staining in the DG. Scale bar = 100 µm. Mean +/− SEM. *p=0.027, **p=0.041, ***p=0.028, ***p=0.006. 1-way ANOVA with Dunnett’s multiple comparison’s test.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Membrane, Expressing, MANN-WHITNEY, Functional Assay, Isolation, Injection, Control, Staining

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Schematic of experimental design. n=10 mice treated with YMP,11 mice treated with AMP. (b) Representative confocal images (left) and quantification (right) of VCAM1+lectin+ in the DG. Hoechst labels cell nuclei. Arrows indicate VCAM1 + vessels. Scale bar = 100 µm. ****p=0.0001. Two-tailed Student’s t-test. Mean +/− SEM. n=10 mice treated with YMP,11 mice treated with AMP. (c) Top: Histogram plots of CD31+VCAM1+ cells isolated from LPS stimulated young (3-month-old) wildtype mice injected (r.o.) with fluorescently tagged DL488 anti-VCAM1 mAb or IgG-DL488 isotype control 2 hours before sacrifice. This was done to set the gating for VCAM1+CD31+BECs. Bottom: Flow gating and histogram plots of pooled (n=4 mice/plasma treatment), young hippocampi isolated from plasma-injected young mice. To label VCAM1+BECs, mice were injected (r.o.) with fluorescently tagged DL488 anti-VCAM1 mAb 2 hours before sacrifice. (d) Quantification of CD31+VCAM1+cells isolated from (left) healthy cortex (n=4 mice per plasma treatment, individually measured) and (right) 4 technical replicates of hippocampi that are pooled from 4 mice per plasma treatment group. Mean +/− SEM. *p=0.017. Two-tailed Student’s t-test. (e) Representative confocal images and quantification (f) in the DG and SGZ of BrdU+, Sox2+, and GFAP. Scale bar = 100 µm. Purple lines outline the SGZ and arrows indicate proliferating NPCs. **p=0.009, *p=0.028. Two-tailed Student’s t-test. Mean +/− SEM. n=10 mice treated with YMP,11 mice treated with AMP. (g) Representative confocal images and quantification (h) in the GCL of DCX (white). Scale bar = 100 µm. ***p=0.0001. Two-tailed Student’s t-test. Mean +/− SEM. n=10 mice treated with YMP,11 mice treated with AMP. (i) Representative confocal images and quantification (j) in the DG of CD68, Iba1, and Hoechst. Scale bar = 100 µm. ***p=0.0047, **p=0.0011, *p=0.031. Two-tailed Student’s t-test. Mean +/− SEM. n=10 mice treated with YMP,11 mice treated with AMP.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Two Tailed Test, Isolation, Injection, Control, Clinical Proteomics

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Violin plots of classical arterial (top) or venous (bottom) markers in each cluster. Putative neurogenic secreted factors include Jag1 and Efnb2. Minima, maxima, median, and percentiles are listed in . (n=146 Capillary BECs, n=59 Venous BECs, n=67 Arterial BECs pooled from 8 mice hippocampi). (b) Violin plots of various angiogenesis and Notch-signaling related genes in each of the 3 distinct clusters. Putative neurogenic secreted factors include Vegfc. Minima, maxima, median, and percentiles are listed in . (n=146 Capillary BECs, n=59 Venous BECs, n=67 Arterial BECs pooled from 8 mice hippocampi). (c) Representative images of Bend.3 cells immunostained for BBB specific markers of adherens junctions (AJ) and tight junctions (TJ), specifically ß-catenin, Claudin-5, and VE-Cadherin. All Bend.3 cells and primary BECs are validated with these markers prior to experimentation; confirmed independently >10 experiments. Hoechst labels cell nuclei. Scale bar = 100 μm. (d) Dose response graph depicting cultured Bend.3 cells stimulated overnight with increasing concentrations of recombinant mouse TNF-α followed by flow cytometry to quantify %CD31+ VCAM1+ cells. n=2 pooled samples per condition. (e) CD31+VCAM1+ Quantification (left) and histogram (right) of Bend.3 cells stimulated overnight with recombinant mouse TNF-α, IL-1β, or IL-6 followed by flow cytometry to measure VCAM1. n=3 biologically independent samples per condition. ****p<0.0001, One-way ANOVA with Tukey’s post hoc test for group comparisons; Mean +/− SEM; experiment repeated four times independently with similar results. (f) Primary BECs and Bend.3 cells cultured in 10% young or aged mouse plasma (YMP: 3-month old; AMP: 18-month-old) or young or aged human plasma (<25 years or >65 years, YHP/AHP) for 16 hours then stained for VCAM1 to label cell nuclei. Representative images are shown. Scale bar = 100 μm. Each plasma treatment experiment in Primary BECs or Bend.3 cells with mouse or human plasma repeated at least three times independently with similar results. (g) Quantification of VCAM1 %area staining. Primary BECs treated with YMP or AMP: n=7 YMP, 9 AMP biologically independent replicates pooled from two experiments. *p=0.0343. Bend.3 cells with YMP or AMP: n=4 biologically independent replicates per group derived from different cell flasks. ***p=0.0003. Bend.3 cells with YHP or AHP: n=6 biologically independent replicates derived from different cell flasks per group. ****p<0.0001. Two-tailed Student’s t-test. Mean +/− SEM. Mean +/− SEM. (h) Bend.3 cells cultured in 10% young or aged mouse plasma (YMP/AMP) for 16 hours followed by flow cytometry of CD31 and VCAM1. n=5 biologically independent replicates per group. Graph of %CD31+VCAM1+ quantification shown with histogram of Bend.3 cells. **p= 0.0082. Two-tailed Student’s t-test. Mean +/− SEM. (i) Quantification of %CD31+ Bend.3 cells treated with young or aged mouse plasma and co-stained with CD31 and ICAM1, E-Selectin, or P-Selectin. n=5 biologically independent replicates per group for ICAM1; n=6 biologically independent replicates per group for E- and P- selectin. Mean +/− SEM. Histogram plots shown to the right of quantifications. Two-tailed Student’s t-test. Not significant; p=0.2355 (ICAM1), p=0.1959 (E-Selectin), p=0.0825 (P-Selectin). (j) Representative images of ICAM1, Meca99, lectin, and Hoechst to label cell nuclei of young (3-month-old) mice which received 7 r.o. injections of young (3 month) or aged (18 month) pooled plasma over 4 days as described in schematic. n=10 mice treated with YMP,11 mice treated with AMP. Scale bar = 100 μm. Quantification (k) on the right using n=4 mice per group. Mean +/− SEM. Two-tailed Student’s t-test. Not significant; p=0.5222. (l-m) Quantification in the DG of total BrdU+Sox2+ neural progenitor cells in young (3-month-old) mice injected r.o. daily over 5 days (2 μg per injection) with TNF-α (n=4 mice/group) or with 3 LPS injections (0.5 mg/kg i.p.) at 28 hours, 22 hours, and 2 hours prior to perfusion (n= 8 mice per group). In each experiment, mice were pulsed with BrdU every 8 hours for 3 injections prior to perfusion. *p=0.0194 (TNF-α *p= 0.0122 (LPS). Mean +/− SEM. Two-tailed Student’s t-test.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Cell Culture, Recombinant, Flow Cytometry, Clinical Proteomics, Staining, Derivative Assay, Two Tailed Test, Injection

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Schematic. n= 5 mice/group. (b) Quantification in the DG of VCAM1 from immunostained confocal images. n= 5 mice/group. Unpaired two-tailed Student’s t-test. Mean +/− SEM. *p=0.0451. (c) Quantification in the DG of BrdU+ and Sox2+ NPCs and triple labeled GFAP+ neural stem cells from confocal images of immunostained sections. Scale bar = 100 μm. n= 5 mice/group. Unpaired two-tailed Student’s t-test. Mean +/− SEM. ***p=0.007, **p=0.0227, *p=0.0038. (d) Quantification in the DG of Iba1 and CD68 from confocal images of immunostained sections. n= 5 mice/group. Unpaired two-tailed Student’s t-test. Mean +/− SEM. *p= 0.0454. (e) Experimental Design. n= 6 Cre− and 7 Cre+ mice per group. (f) Quantification of VCAM1+ percent area in lectin+ vasculature of immunostained sections from 6 Cre− and 5 Cre+ mice/group. ****p<0.0001. Unpaired two-tailed Student’s t-test. Mean +/− SEM. (g) Quantification of the total number of BrdU+ cells, BrdU+Sox2+ co-labeled neural progenitor cells, and (h) average # DCX+ imature neurons per section in the DG of immunostained sections. n=6 Cre− and 7 Cre+ mice per group. *p=0.0012, **p=0021, ***p=0.0028. Unpaired two-tailed Student’s t-test. Mean +/− SEM. (i) Quantification of Iba1 and CD68 in the DG of immunostained sections. n=6 Cre− and 7 Cre+ mice per group. Bars represent mean. Error bar represents SEM. Stain experiment repeated twice with similar results; Similar mouse experiments using these validated transgenic mice repeated 4 times with similar results (see ). (j) Confocal images of brain sections of Cre+ or Cre− aged Slco1c1-CreERT2-Vcam1fl/fl mice treated with tamoxifen in young adulthood (age 2 months) and aged to 18 months stained for anti-VCAM1 or IgG isotype control, Aqp4, and GFAP. Hoechst labels cell nuclei. Aged (18-month-old) brain sections were treated with Sudan Black B to remove lipofuscin background in the granular and hilus layers of the DG. SBB treatment removes the majority of lipid-based artifacts typically seen in aged tissues without suppressing immunofluorescent labeling. Scale bar = 100 μm. Experiment repeated three times with similar results. (k) Aged (18-month-old) Cre+ and Cre− brain sections were immunostained using the regular protocol, without Sudan Black B treatment. Heavy lipofuscin background is present in the Cy3 fluorescence channel. Experiment repeated three times with similar results.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Two Tailed Test, Labeling, Staining, Transgenic Assay, Control, Fluorescence

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Representative 2D and 3D Z-stacked high magnification confocal images (51 slices with an interval of 0.4 um) of VCAM1 in the granular layer of the DG of the hippocampus of a young (3-month-old) NSG mouse acutely treated with Aged Human Plasma (AHP). Brain sections were co-stained with DCX and NeuN to label immature and mature granule neurons, respectively. VCAM1 is not expressed in these cell types. Light blue lines outline the granule layer. Experiment repeated 3 times independently with similar results. 2D Scale bar = 50 μm. Two 3D renderings of the 2D images are displayed. 3D Scale bar = 20 μm. (b) Quantification of VCAM1, Aqp4, Lectin, with Hoechst labeling cell nuclei in the hippocampus and cerebellum of EAE (multiple sclerosis), Npc1−/− (Niemann Pick Disease Type C), and Grn−/− (Frontotemporal Dementia) disease models. EAE: n=4 naïve, 8 EAE induced, *p=0.006, **p=0.0125; Npc1: n=6 mice per group, ***p=0.0274, ****p<0.0001; Grn: n=4 mice per group, *****p=0.0004. Unpaired two-tailed Student’s t-test. Mean +/− SEM. (c) Representative 2D and 3D Z-stacked high magnification confocal images (51 slices with an interval of 0.4 um) of VCAM1 in te granular layer of the DG of the hippocampus co-stained with Sox2 and GFAP to label neural stem and progenitor cells (Sox2+GFAP+) and hilus GFAP+ astrocytes. VCAM1 is not expressed in these cell types in the DG. Light blue lines outline the granule layer. Experiment repeated 3 times independently with similar results. 2D Scale bar = 50 μm. Two 3D renderings of the 2D images are displayed. 3D Scale bar = 20 μm. (d) Vcam1fl/flSlco1c1-CreERT2+/− (Cre+) or CreERT2−/− (Cre−) littermates (3-month-old) were treated daily with tamoxifen (i.p. 150 mg/kg) for 5 days followed by 4 days of rest. Mice received 3 LPS injections (0.5 mg/kg i.p.) at 28, 22, and 2 hours prior to perfusion. Mice also received a retro-orbital injection of fluorescently conjugated mouse anti-VCAM1 mAb (100μg) 2 hours prior to perfusion. FACS gating strategy to analyze single BECs. PI+ dead cells were excluded. CD11a/b, CD45, and Ter-119 negative cells were gated to exclude erythrocytes, monocytes/macrophages and microglia. CD13 and ACSA-2 staining was applied to exclude pericytes and astrocytes, respectively. CD31+MECA99+ cells were defined the BEC population. (e) Quantification of (f) flow cytometry that was performed on primary BECs isolated from Cre+ or Cre− mice treated as described in (d). n=3 Cre+ or Cre− mice received LPS, while one Cre− mouse was given PBS vehicle control instead. The VCAM1 gate was set based on a Cre− mice injected with fluorescently conjugated IgG. **p=0.0011; Unpaired two-tailed Student’s t-test; Mean +/− SEM. (g) Representative confocal images of cortex and DG for VCAM1 and Hoechst to label cell nuclei in LPS stimulated mice as descrbed in (d). Loss of Vcam1 in Cre+ mice, but not Cre−, in BBB endothelium, but not in meninges is shown. Experiment repeated 3 times independently with similar results. Scale bar = 100 μm.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Clinical Proteomics, Staining, Labeling, Two Tailed Test, Injection, Flow Cytometry, Isolation, Control

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Experimental design. n=7 Cre− mice administered YMP, 8 Cre+ mice administered YMP, 8 Cre− mice administered AMP, 8 Cre+ mice administered AMP. Plasma administration in these transgenic mice was performed 1 additional time in a long-term paradigm with similar results . Plasma administration was performed in 8 independent experiments with similar results . (b) Representative confocal images in the DG of VCAM1, MECA-99, and Aqp4. Hoechst labels cell nuclei. Scale bar = 200 µm for merged images and scale bar= 50 µm for the zoomed VCAM1 and MECA-99 merged images outlined with white squares. Tissue was stained and VCAM1 was measured in all 31 mice in this study. (c) Quantification of VCAM1+ lectin+ vasculature ***p=0.0031. 2-way ANOVA with Tukey’s multiple comparisons test. Mean +/− SEM. n=7 Cre− mice administered YMP, 8 Cre+ mice administered YMP, 8 Cre− mice administered AMP, 8 Cre+ mice administered AMP. (d) Mouse sVCAsM1 ELISA of plasma samples. *p=0.022. 2-way ANOVA with Tukey’s multiple comparisons test. Mean +/− SEM. n=7 Cre− mice administered YMP, 8 Cre+ mice administered YMP, 8 Cre− mice administered AMP, 8 Cre+ mice administered AMP. (e) BrdU quantification and representative confocal images (f) and BrdU+Sox2+ quantification (g) in the DG of brain sections immunostained for BrdU and Sox2. White dotted lines outline the SGZ; Scale bar = 200 µm. *p=0.02, **p=0.017. 2-way ANOVA with Tukey’s multiple comparisons test. Mean +/− SEM. n=7 Cre− mice administered YMP, 8 Cre+ mice administered YMP, 8 Cre− mice administered AMP, 8 Cre+ mice administered AMP. (h) DCX+ quantification and representative confocal images (i) in the GCL. Hoechst labels cell nuclei. Scale bar = 100 µm. **p=0.0015. 2-way ANOVA with Tukey’s multiple comparisons test. Mean +/− SEM. n=7 Cre− mice administered YMP, 8 Cre+ mice administered YMP, 8 Cre− mice administered AMP, 8 Cre+ mice administered AMP. (j) Representative confocal images and quantification (k) from the DG of CD68 and Iba1. Hoechst labels cell nuclei. Scale bar = 100 µm. ****p=0.0008, ***p=0.0061, 2-way ANOVA with Tukey’s multiple comparisons test. Mean +/− SEM. n=7 Cre− mice administered YMP, 8 Cre+ mice administered YMP, 8 Cre− mice administered AMP, 8 Cre+ mice administered AMP.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Clinical Proteomics, Transgenic Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Experimental design. n=7 mice/group. (b) Ponceau S stain showing total protein pull-down from plasma by both IgG and anti-VCAM1 mAb conjugated beads. Experiment repeated 3 times with similar results. (c) Western blot showing human sVCAM1 (93 kDa) pulled down during immunodepletion by anti-human VCAM1 antibody but not IgG. Experiment repeated 3 times with similar results. Full blots shown in Source Data. (d) Human sVCAM1 ELISA of depleted plasma. n=4 mice per group. Mean +/− SEM. (e) Representative confocal images and quantification (f) in the DG of VCAM1, MECA-99, and Aqp4. Hoechst labels cell nuclei. Scale bar = 50 μm for merged images and scale bar= 20 μm for the 4x zoomed single channel VCAM1 images outlined with white squares. Arrows indicate VCAM1+ vessels. n=5 mice/group analyzed. Mean +/− SEM. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. ***p=0.0004, **p= 0.0025. (g) Quantification of the total number of BrdU+ and BrdU+Sox2+ co-labeled neural progenitor cells in the DG of immunostained sections. n=7 mice/group. Mean +/− SEM. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. *p=0.0237, **p= 0.0123, ***p=0.0320, ****p=0.0094. (h) Quantification and representative confocal images (i) of the DG for DCX and Hoechst to label cell nuclei. Scale bar = 100 μm. n= 5 mice/group analyzed. Mean +/− SEM. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. **p=0.0017, *p=0.0385. (j) Quantification of the Iba1+ and CD68+ staining from confocal images in the DG. n=7 mice/group. *p= 0.0156, **p=0.0242, ***p=0.0034, ****p= 0.0237, p=0.0546 PBS compared to anti-VCAM1 activated microglia counts. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. Mean +/− SEM. (k) Experiment schematic. n=9 PBS-treated, 8 AHP + IgG-treated, and 8 AHP + anti-VCAM1 mAb-treated mice. (l) Quantification in the DG of VCAM1 in lectin+ blood vessels using immunostained confocal images. n=9 PBS-treated, 8 AHP + IgG-treated, and 7 AHP + anti-VCAM1 mAb-treated mice analyzed. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. Mean +/− SEM. **p=0.006. (m) Quantification in the DG of CD68 in Iba1+ stained microglia using immunostained confocal images. n=9 PBS-treated, 8 AHP + IgG-treated, and 8 AHP + anti-VCAM1 mAb-treated mice. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. Mean +/− SEM. ***p=0.0006, **p=0.0067. (n) Quantification of BrdU+Sox2+ progenitor cells and DCX+ immature neurons (o) from confocal images. n=9 PBS-treated, 8 AHP + IgG-treated, and 8 AHP + anti-VCAM1 mAb-treated mice. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. Mean +/− SEM. *p=0.018, **p=0.0386, ***p=0.0344, ****p=0.0167. (p) Quantification of the total numbers of EdU+ surviving cells in the DG of immunostained sections. n=9 PBS-treated, 8 AHP + IgG-treated, and 8 AHP + anti-VCAM1 mAb-treated mice. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. Mean +/− SEM. ***p=0.0009, ****p=0.0002.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Staining, Clinical Proteomics, Western Blot, Immunodepletion, Enzyme-linked Immunosorbent Assay, Labeling

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Experimental Design. n=8 mice per group. (b) Representative confocal images and (c) quantification in the DG of VCAM1, MECA-99, and Aqp4. Hoechst labels cell nuclei. Scale bar = 100 μm. Arrows point to VCAM1+ vessels. (n=4 mice/group analyzed). 2-way ANOVA with Tukey’s multiple comparisons test. Mean +/− SEM.***p=0.0002. (d-f) Quantification of the total number of BrdU+ cells, BrdU+Sox2+ neural progenitor cells, and DCX+ immature neurons in the DG of immunostained sections. n=8 mice per group. 2-way ANOVA with Tukey’s multiple comparisons test. Mean +/− SEM. *p=0.0193, **p=0.0283, ***p=0.0015. (g-h) Quantification of the total number of surviving EdU+DCX+ immature neurons and EdU+NeuN+ neurons in the DG of immunostained sections. n=8 mice per group. 2-way ANOVA with Tukey’s multiple comparisons test. Mean +/− SEM. *p=0.0181. (i-j) Quantification of Iba1 and CD68 in the DG of immunostained sections. n=8 mice per group. 2-way ANOVA with Tukey’s post-hoc test. Mean +/− SEM.****p<0.0001 for both. (k) Schematic. Aged (18-month-old) C57BL6/J female mice received i.p. injections of a mouse specific anti-VCAM1 mAb or IgG isotype control (9 mg/kg) every 3 days for a total of 7 injections. Mice also received BrdU daily (100 mg/kg i.p.) for 6 consecutive days followed by perfusion 2 days after the last injection. n=9 IgG- treated and 10 anti-VCAM1 mAb-treated mice per group. (l) Quantification of VCAM1+Lectin+ staining from confocal images in the DG. n=3 mice brain sections stained and quantified per group. Mean +/− SEM. *p=0.0128, 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. (m) Quantification of BrdU+ and BrdU+Sox2+ staining from confocal images in the DG. n=9 IgG- treated and 10 anti-VCAM1 mAb-treated mice per group. Unpaired two-tailed Student’s t-test. Mean +/− SEM. *p=0.0325, ***p=0.0003. (n) Quantification of Iba1 and CD68 staining from confocal images in the DG. n=9 IgG- treated and 10 anti-VCAM1 mAb-treated mice per group. **p=0.0008, *p= 0.0427. Unpaired two-tailed Student’s t-test. Mean +/− SEM. (o) sVCAM1 ELISA of the plasma of young (4-month-old) and aged (18-month-old) female mice. n=6 mice/group. ****p<0.0001. Unpaird two-tailed Student’s t-test. Mean +/− SEM.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Control, Injection, Staining, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Clinical Proteomics

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Experimental design. n=10 mice per group. (b) Representative confocal images and quantification (e) (n= 5 mice/group) in the DG of VCAM1, lectin, and Aqp4. Hoechst labels cell nuclei. White arrows point to VCAM1+ vessels. Scale bar = 100 µm. Mean +/− SEM. 2-way ANOVA with Tukey’s multiple comparisons test. ****p=0.0013, p=0.06 (PBS vs. AHP in mice treated with anti-VCAM1 mAb). (c) Representative confocal images and quantification (f) in the DG of EdU and Sox2. Hoechst labels cell nuclei. Arrows indicate proliferating NPCs. The SGZ is outlined with white lines. Scale bar = 50 µm. n=10 mice/group. Mean +/− SEM. 2-way ANOVA with Tukey’s multiple comparisons test. *p=0.0154. (d) Representative confocal images and quantification (g) in the DG of CD68 and Iba1. Hoechst labels cell nuclei. Scale bar = 100 µm. n=10 mice/group. Mean +/− SEM. 2-way ANOVA with Tukey’s multiple comparisons test. ****p<0.0001, ***p=0.0001, *p=0.0407. (h) Experimental design. n=8 mice injected with PBS (r.o.), 8 mice injected with AHP (r.o.) and IgG (i.p.), and 7 mice injected with AHP (r.o.) and anti-VCAM1 mAb (i.p.) (i) Quantification and (j) representative confocal images in the DG of BrdU+ and BrdU+Sox2+ precursor cells. The SGZ is outlined with white lines. Scale bar = 100 µm. n=8 mice injected with PBS, 8 mice injected with AHP and IgG, and 7 mice injected with AHP and anti-VCAM1 mAb. Mean +/− SEM. One-way ANOVA with Tukey’s post hoc test for group comparisons. **p=0.0056, *p=0.0253, ***p=0.0041, ****p=0.019. (k) Quantification in the DG of total number of surviving EdU+ cells, EdU+GFAP+ astrocytes, and EdU+Sox2+GFAP+ radial glia-like NSCs in the SGZ based on confocal images of immunostained brain sections for EdU, Sox2, and GFAP. n=8 mice injected with PBS, 8 mice injected with AHP and IgG, and 7 mice injected with AHP and anti-VCAM1 mAb. Mean +/− SEM. One-way ANOVA with Tukey’s post hoc test for group comparisons. **p=0.011, **p=0.0057, ***p=0.0083, ****p=0.049, *****p=0.022. (l) Representative confocal images and quantification (m) in the GCL of EdU, DCX, and NeuN. Scale bar = 100 µm. n=8 mice injected with PBS, 8 mice injected with AHP and IgG, and 7 mice injected with AHP and anti-VCAM1 mAb. Mean +/− SEM. One-way ANOVA with Tukey’s post hoc test for group comparisons. *p=0.0399 and p=0.0643(AMP+IgG vs. AMP+ anti-VCAM1 mAb). (n) Cell fate based on co-labeling of surviving EdU+ cells 4 weeks after EdU labeling of mice. Each bar represents 100% of EdU+ cells.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Injection, Labeling

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Quantification of fluorescent signal measured with a microplate reader from homogenized brain tissues samples from mice that were injected with Texas Red labeled 70kDa dextran r.o. and perfused with FITC labeled 2MDa dextran 3 hours after injection. Vcam1-fl/fl Slco1c1-CreERT2−/− (Cre−) or Vcam1-;fl/fl Slco1c1-CreERT2−/+ (Cre+) mice were used. n= 3 Young Cre− (5-month-old), 5 aged Cre− (19-month-old), 2 young Cre− mice that underwent TBI as a positive control, 3 young Cre− control mice not injected with dextran, and 3 aged Cre− control mice not injected with dextran. Mean +/− SEM. (b) Quantification of fluorescent signal from homogenized brain tissues samples measured with a microplate reader. Cre− or Cre+ mice were used as described in (a). n= 3 Young Cre−, 5 aged Cre−, or 5 “Aged Vcam1-ST” (19-month-old), which are Cre+ mice that were tamoxifen treated for 4 days, 2 months prior to sacrifice, and that were infused with dextran prior to sacrifice as described in (a). Mean +/− SEM. (c-d) Quantification of Mean Fluorescence Intensity from confocal images of tissue sections from mice injected as in (a-b). Cre− or Cre+ mice were used as described in (a). n= 4 Young Cre−, 5 aged Cre−, 5 “Aged Vcam1-ST” Cre+ mice that were tamoxifen treated and that were infused with dextran as described in (a-b), 1 young and 1 aged Cre− control mice not infused with dextran. Mean +/− SEM. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test. p=0.895 (Young vs. Aged), 0.9097 (Aged vs. Aged Vcam1-ST). (e) Schematic of flow cytometric analysis of various immune cell populations from mouse cortex and hippocampi. (f) Flow cytometry gating strategy of individual hippocampal immune cell populations labeled with various immune cell markers, anti-alpha4 and anti-beta1 integrins (VLA-4). n-1 was used to gate fir VLA-4+ cell populations.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Injection, Labeling, Positive Control, Control, Fluorescence, Flow Cytometry

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Mouse model and experimental groups. n= 5 Young Cre− mice, 9 Aged Cre− mice, 4 “Vcam1− (LT)” mice, and 4 “Vcam1-(ST)” mice. Vcam1-fl/fl Slco1c1-CreERT2−/− (Cre−) or Vcam1-fl/fl Slco1c1-CreERT2−/+ (Cre+) mice were used. Tamoxifen treatment paradigm described in schematic. (b) Gating plots of CD31+VCAM1+ hippocampal and cortex cells isolated from 1 LPS stimulated aged (19-month-old) Cre+ (Vcam1−deletion ST) mouse and 1 Cre− mouse injected with fluorescently tagged DL488 anti-VCAM1 mAb (r.o.) 2 hours before sacrifice to confirm VCAM1 on BECs was reduced. 1 additional Cre− mouse was treated with LPS and injected with IgG-DL488 isotype control prior to sacrifice to serve as a control for VCAM1 gating. (c-j) Quantification of various cell populations present in Young Cre− (n=5), Aged Cre− (n=9), Aged Vcam1-deleted LT (n=4), and Aged Vcam1-deleted ST (n=4) mice per group. Mean +/− SEM. *p=0.0413, **p=0.0245, ***p=0.0429, ****p=0.0023. Mean +/− SEM. 1-way ANOVA with Tukey’s multiple comparisons post-hoc test.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Isolation, Injection, Control

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Experimental design for anti-VLA-4. n=7 mice/group. (b) Representative confocal images and quantification (d) of VCAM1, Lectin, and Hoechst to label cell nuclei. Scale bar = 100 μm. n=3 mice/group analyzed. Mean +/−SEM. (c) Representative confocal images and quantification (f) in the DG of CD68, Iba1, and Hoechst. Scale bar = 100 μm. n=7 mice/group. Mean +/−SEM. Two-tailed Student’s t-test. *p=0.0436, **p=0.0175. (e) Quantification of confocal images of the DG of NPCs co-labeled with BrdU and Sox2. n=7 mice/group. Mean +/−SEM. (g-h) Quantification of Iba1+ and Iba1+CD68+ Microglia in the DG from the experiment described in . n=8 mice/group. Mean +/−SEM. (i-k) 13-month-old NSG mice were injected with anti-VCAM1 mAb or IgG every 3 days for one month and underwent novel object recognition or fear conditioning during the last week (n=11 mice per group). Quantification of percent time spent exploring objects in novel object placement task is shown in (i) while %Freezing observed during the Training (j) phase is shown. The average of Trials 3-5 for Contextual are quantified in (k). Mean +/−SEM. 2-way Anova with Sidak’s multiple comparisons test. *p=0.0485. There were no significant differences between groups for contextual freezing (Two-tailed Student’s t-test; p=0.2722). (l-n) 23-month-old C57BL6 mice were injected with anti-VCAM1 or IgG every 3 days for one month and underwent fear conditioning during the last week (n=7 PBS, 12 IgG, and 13 anti-Vcam1-treated mice per group). %Freezing observed during the Training (l), Cued (m), and Contextual (n) tests are shown. Mean +/− SEM. 2-way Anova with Tukey’s multiple comparisons test between groups at each timepoint. *p=0.0493. Individual data point distribution shown in Source Data.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Two Tailed Test, Labeling, Injection

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: (a) Experimental design for anti-VCAM1. n=7 mice/group. (b) Representative confocal images of BrdU and Sox2 from the experiment described in . Arrows indicate proliferating NPCs. The white lines outline the SGZ. Scale bar = 100 µm. n=7 mice/group. (c) Representative confocal images of CD68, Iba1, and Hoechst from the experiment described in . Scale bar = 100 µm. n=7 mice/group. (d) Quantification in the DG of BrdU and Sox2. n=7 mice/group. Two-tailed Student’s t-test. Mean +/− SEM. *p=0.0341, **p=0.0027. (e) Quantification in the DG of CD68 and Iba1 from confocal images. n=7 mice/group. Mean +/− SEM. Two-tailed Student’s t-test. ***p=0.0005, **p=0.0026, *p=0.0354. (f) Experimental design for conditional deletion of Vcam1 in young (2-month-old) mice followed by aging them to 18 months. n=8 mice/group. (g) Quantification of total BrdU+ proliferating cells, and BrdU+Sox2+ neural progenitor cells in the DG of immunostained sections. n=8 mice/group. Mean +/−SEM. Two-tailed Student’s t-test. **p=0.0075, *p=0.0263. (h) Quantification in the DG of CD68 and Iba1. Hoechst labels cell nuclei. n=8 mice/group. Mean +/−SEM. Two-tailed Student’s t-test. **p=0.0068, *p=0.0169. (i) Days 1–5 escape latency from Barnes Maze and (j) percent time spent exploring objects in novel object placement task of IgG treated young adult C57BL6 mice (5-month-old; n=15) and IgG-treated aged mice (17-month-old; n=15) or anti-VCAM1 mAb treated aged mice (17-month-old; n=15). All mice received intraperitoneal injections every 3 days for 3 weeks prior to initiating behavior studies and throughout the duration of the studies; two-way repeated-measures ANOVA with Bonferroni’s post hoc test for time × group comparisons; One-way ANOVA with Tukey’s post hoc test for group comparisons; *p=0.0217, **p<0.01, ***p<0.001; ****p<0.0001; Mean +/− SEM. (k) Quantification of freezing behavior in Fear Conditioning Contextual trial with 23-month-old C57BL6 mice injected with anti-VCAM1 mAb or IgG every 3 days for one month. Average of trials 3–5 shown. n=7 PBS, n=12 IgG, n= 13 anti-Vcam1−treated mice per group. **p=0.0075, *p=0.0265. One-way ANOVA with Tukey’s post hoc test for group comparisons; Mean +/− SEM.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Two Tailed Test, Injection

Journal: Nature medicine

Article Title: Aged blood impairs hippocampal neural precursor activity and activates microglia via brain endothelial cell VCAM1

doi: 10.1038/s41591-019-0440-4

Figure Lengend Snippet: In young healthy mice, neurovascular homeostasis is maintained with low expression levels of systemic soluble VCAM1 (sVCAM1) and BBB-specific VCAM1, active neurogenesis with neural stem cells (NSCs) differentiating into NPCs (NPCs), immature neurons and mature neurons, and nonreactive microglia in a low inflammation environment. During aging or exposure to aged plasma, we propose: 1) Inflammatory factors in aged plasma (IL-1β, TNF-α, among others) induce arterial and venous BEC activation and upregulation of VCAM1 through their cytokine receptors Tnfrsf1a and Il1r1. 2) Venous VCAM1 facilitates tethering, but not transmigration, of leukocytes which sustain BEC inflammation. 3) Inflamed and activated venous and arterial VCAM1+ brain endothelium relay (unknown) signals to the parenchyma leading to a loss of homeostasis, decline in NPC activity and chronic activation of microglia. 4) anti-VCAM1 mAb protects young brains from the detrimental effects of aged plasma by reducing BEC-mediated inflammation. 5) anti-VCAM1 mAb rejuvenates aged brains by reducing BEC-mediated inflammation and VCAM1+ BEC-mediated reduction in NPC proliferation.

Article Snippet: Rat monoclonal anti-BrdU (1:500, Abcam, ab6326, clone BU1/75[ICR1]) Rat monoclonal anti-VCAM1 (1:125, Abcam, ab19569, clone M/K-2) Goat monoclonal anti-Sox2 (1:100, Santa Cruz, sc-17320, clone Y-17) Goat polyclonal anti-doublecortin (DCX) (1:100, Santa Cruz, sc-8066, clone C-18) Goat polyclonal VE-Cadherin (Santa Cruz Biotechnology, sc-6458, clone C-19) Click-iT® Plus EdU Alexa Fluor® 488 Imaging Kit (Thermo/Life Technologies, C-10637) Mouse monoclonal anti-GFAP (1:1000, Chemicon/Fisher, MAB360MI, clone GA5) DyLight 488 Lectin (1:200, Vector, DL-1174) Rabbit monoclonal anti-Aquaporin 4 (1:500, Millipore, AB2218, clone) Rat monoclonal anti-CD68 (1:600, Serotec, MCA1957, clone FA-11) Rabbit polyclonal anti-Iba1 (1:250, ProteinTech, 10904–1-AP) Mouse anti-human-VCAM1 antibody (Novus Biologicals, BBA5, clone BBIG-V1) Mouse monoclonal anti-human IgG antibody (R&D Systems, MAB002 clone 11711) Rat monoclonal anti-VCAM-1 (BioxCell, BE0027, clone M/K-2.7) Rat IgG1 Isotype antibody (BioxCell, BE0088, clone HRPN)

Techniques: Expressing, Clinical Proteomics, Activation Assay, Transmigration Assay, Activity Assay

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Immunofluorescence imaging of immune synapses between H1299 lung cancer cells and γδ T cells by phosphotyrosine (pTyr) staining. H1299 lung cancer cells are pretreated with phosphate- buffered saline (PBS) or DAC prior to coculture with γδ T cells. Quantifications of immune synapses per cancer cell on eight randomly taken high power fields for each treatment are shown in the dot plots (mean ± SD). Scale bar: 100 μm. p value is calculated by the Mann-Whitney test. ( B ) A scatter plot of DAC-induced surface proteomes in H1299 (y-axis) and A549 (x-axis) human lung cancer cells following daily treatment of 100 nM DAC for 72 hours and culture in drug-free medium for 3 days (D3R3). ICAM-1 is among the top upregulated surface proteins by DAC in both cells. ( C ) Western blot analyses of ICAM-1 protein expression in mock-treated vs. DAC-treated human lung cancer cells. D3: daily treatment of 100 nM decitabine for 72 hours. D3R3: daily treatment for 72 hours, followed by a 3-day rest period in drug-free medium. β-actin: loading control. ( D ) Immunofluorescence staining of ICAM-1 and immune synapse molecules (e.g., LFA-1, LAT) at immune synapses formed between γδ T cells and DAC-treated H1299 lung cancer cells. Scale bar: 10 μm. ( E ) Representative flow cytometric dot plot showing H1299 lung cancer cells with CRISPR-knockout of ICAM1 (KO-ICAM1) subject to γδ T cell killing for 2 hours. The effector to target (E: T) ratio is 3:1. Lung cancer cells are pre-treated with mock, DAC alone, γδ T cells alone or a combination of DAC and γδ T cells. The X-axis denotes surface ICAM1 levels. Y-axis represents signal intensities of propidium iodide. ( F ) Bar graphs showing percent cell death of human lung cancer cell lines (i.e., H1299, CL1-0, and A549) with CRISPR-knockout of ICAM-1 subject to γδ T cell killing for 2 hours. Cell death is measured by Annexin V and propidium iodide apoptosis assays (mean ± SEM, n = 3). Statistical significance is determined by one-way ANOVA test. ( G ) Representative flow cytometric dot plot showing H1299 lung cancer cells with a Tet-on expression system of ICAM1 (OV-ICAM1) subject to γδ T cell killing for 2 hours. Doxycycline (1 μg/mL) is added 24 hours prior to coculture to induce ICAM-1 protein expression. Cell death is measured by Annexin V (x-axis) and propidium iodide (y-axis) apoptosis assays. ( H ) Bar graphs showing cell death of human lung cancer cell lines (i.e., H1299, CL1-0, and A549) with ICAM-1 over-expression subject to γδ T cell killing for 2 hours. E:T ratio is 3:1. Cell death is measured by Annexin V and propidium iodide apoptosis assays. Statistical significance is determined by one-way ANOVA test (* p < 0.05, ** p < 0.01, ***, p < 0.001). ( I ) Immunofluorescence imaging of immune synapses between H1299 KO-ICAM1 lung cancer cells and γδ T cells by phosphotyrosine (pTyr) staining. Scale bar: 100 μm. Quantifications of immune synapses per cancer cell on six randomly taken high power fields for each treatment are shown in the dot plots (mean ± SD). p value is calculated by the Mann-Whitney test.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Immunofluorescence, Imaging, Staining, Saline, MANN-WHITNEY, Western Blot, Expressing, Control, CRISPR, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: Sequencing results of the KO-ICAM1 lung cancer cells are aligned against the reference sequence of the ICAM1 genome locus. Alignment gaps are denoted as hyphens (-) to mark the lost (knockout) regions of the edited ICAM1 genome locus.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Sequencing, Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Immunofluorescence staining of F-actin (red), ICAM-1 (green), pTyr (phosphotyrosine, white) at immune synapses between γδ T cells and DAC-pretreated H1299 lung cancer cells at D3R3. Accumulation of F-actin beneath the cell membrane is noted in DAC-pretreated lung cancer cells. DAPI: 4′,6-diamidino-2-phenylindole, as a nuclear counterstain. Scale bar: 10 μm. ( B ) Representative immunofluorescence images of the interfaces between γδ T cells and H1299 lung cancer cells (parental vs. ICAM-1 knockout (KO-ICAM1)). Signals of F-actin (red) in the periphery of H1299 cancer cells are shown in two- and-a-half-dimensional (2.5D) images in the lower panels. Scale bar: 10 μm. ( C ) Dot plots of signal intensities of F-actin (left panel) and ICAM-1 (right panel) from five pTry-positive immune synapses between γδ T cells and H1299 lung cancer cells (parental or KO-ICAM1). p value is calculated by two-way ANOVA test. ( D ) Immunofluorescence images of immune synapses between γδ T cells (marked with T) and H1299 lung cancer cells (marked with C) stained for ICAM-1 (green), F-actin (Red) and phosphotyrosine (pTyr, white). Lung cancer cells (parental or KO-ICAM1) are pretreated with PBS (Mock) or 100 nM DAC and cocultured with γδ T cells at D3R3. ( E ) Dot plots of F-actin signal intensities at immune synapses between γδ T cells and H1299 cells. H1299 cells are pretreated with PBS (Mock), DAC alone or combination of DAC pretreatment (D3R3) and 1 μg/mL Cyto B (cytochalasin B, an inhibitor of actin filament polymerization) for 1.5 hours prior to coculture with γδ T cells (mean ± SD). p value is calculated by one-way ANOVA with Tukey’s multiple comparisons test (***, p < 0.001; ****, p < 0.0001). ( F ) Representative immunofluorescence images of immune synapses (pTyr staining) between γδ T and H1299 cells pretreated with PBS (Mock), DAC alone, and combination of DAC and Cyto B. Blow-up images of the square areas for each treatment are shown in the lower panels. Arrows denote immune synapses between γδ T and H1299 cells. Scale bar: 100 μm (upper) and 20 μm (lower panels). ( G ) Dot plots showing numbers of immune synapses per cancer cell on eight randomly taken high power fields for H1299 cells pretreated with PBS (Mock), DAC, and combination of DAC and Cyto B (mean ± SD). p value is calculated by one-way ANOVA with Tukey’s multiple comparisons test (*, statistical significance).

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Immunofluorescence, Staining, Membrane, Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: Parental or ICAM-1 knockout (KO-ICAM1) H1299 cells are pretreated daily with PBS (Mock) or 100 nM DAC for 72 hours followed by 3-day drug-free culture before coculture with γδ T cells. Signal intensities of each protein (F-actin, red; ICAM-1, green; phosphotyrosine, pTyr, white) along the immune synapse area are graphed on the right. DAPI: 4’,6-diamidino-2-phenylindole, as nuclear counterstain. Scale bar: 10 μm.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Visualization of multi-omics data (i.e., mRNA-seq, Omni-ATAC-seq, and MethylationEPIC arrays) for DAPK3 , EVPLL, and TUBE1 in H1299 lung cancer cells. ( B ) Promoter methylation status and mRNA expression levels of the ICAM1 gene measured by Infinium MethylationEPIC arrays (left panels) and mRNA-seq (right panels) in human lung cancer cells treated without and with DAC 100 nM DAC for 3 days followed by a 3-day drug-free culture. ( C ) Open chromatin regions in the promoter areas of the ICAM1 gene in human lung cancer cells upon 100 nM DAC treatment analyzed by Omni-ATAC-seq. The green bar represents a CpG island. ( D ) Validation of Omni-ATAC-seq by quantitative real-time PCR on transposase-accessible chromatin at the ICAM1 promoter of human lung cancer cells subject to daily treatment of 100 nM DAC treatment for 3 days, followed by a 3-day drug-free culture. Experiments are performed in triplicates, and data are presented as mean ± SD. p value was calculated by unpaired t test (*, p < 0.05). ( E ) IPA Network analysis of mRNA expression changes in human lung cancer cells treated by DAC reveals coordinated changes of the immune-related surface molecules and the cytoskeleton-associated genes. ( F ) IPA upstream regulator analysis of mRNA expression changes in human lung cancer cells treated by DAC. T cell effector cytokines such as TNF-α and IFN-γ may enhance DAC-induced expression changes of immune-related molecules and ICAM-1 in lung cancer cells. TP53 is a potential master regulator for cancer cytoskeleton reorganization essential for DAC-potentiated γδ T cell killing.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Biomarker Discovery, Methylation, Expressing, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Diagram of transcription factor binding sites at the ICAM1 promoter derived from the ENCODE ChIP-seq data ( https://www.encodeproject.org ). Visualizations of ATAC-seq peaks at the ICAM1 promoter in PC9 and CL1-5 lung cancer cell lines subject to DAC treatment are shown above. ( B ) Promoter methylation status and mRNA expression levels of putative transcription factors (i.e., RELB, NFKB2, STATS, and RUNX3) at the ICAM1 promoter in A549, H1299, PC9, and CL1-5 lung cancer cells. Dot and line plots represent methylation levels (β values) of promoter probes measured by Infinium MethylationEPIC arrays. The promoter probes with β values greater or equal to 0.5 at baseline (Mock) are shown. Bar graphs represent relative mRNA expression levels based on normalized FPKM measured by mRNA-seq.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Binding Assay, Derivative Assay, ChIP-sequencing, Methylation, Expressing

Journal: eLife

Article Title: GIPC proteins negatively modulate Plexind1 signaling during vascular development

doi: 10.7554/eLife.30454

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , Non-targeting gRNA1. Pool of HUVEC/TERT2 cells. , This paper , , Derived from HUVEC/TERT 2 cell line (ATCC CRL4053). Cells were grown under blasticidin (4 μg/ml) selection and used between 7th and 10th passages. Cells stably coexpress Cas9 nuclease and non-targeting gRNA1 (from Torres-Vázquez lab plasmid #1859).

Techniques: Transgenic Assay, Plasmid Preparation, Mutagenesis, Derivative Assay, Selection, Stable Transfection, Knock-Out, Recombinant, Control, shRNA, Sequencing, Construct, Synthesized, Positive Control, Sterility, Concentration Assay