MA3105

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Flow cytometry analysis of CD31 in HUVEC cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a CD31 monoclonal antibody (Product # MA3105) at a dilution of 0.5 µg/test for 60 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

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Flow cytometry analysis of CD31 in mouse splenocytes (green) compared to an isotype control (blue). Human blood was collected, combined with a hydrophilic polysaccharide, centrifuged, transferred to a conical tube and washed with PBS. 50 µL of cell solution was added to each tube at a dilution of 2x10^7 cells/mL, followed by the addition of 50 µL of isotype control and primary antibody (Product # MA3105) at a dilution of 0.5 µg/test. Cells were incubated for 30 min at 4ºC and washed with a cell buffer, followed by incubation with a DyLight 488-conjugated secondary antibody for 30 min at 4ºC in the dark. FACS analysis was performed using 400 µL of cell buffer.

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Figure 1 CD93 interacts with MMRN2 in endothelial cells and colocalizes with MMRN2 in tumor vasculature. ( A ) Western blot against MMRN2 in CD93 coimmunoprecipitated samples (CD93 co-IP) derived from total protein lysates of HDMECs. A nonrelevant IgG antibody was used as negative control (IgG co-IP). CD93 FT and IgG FT represent the flow-through of the unbound fraction. ( B ) In situ proximity ligation assay (PLA) for CD93 and MMRN2 in cultured HDBECs. Positive signal (green dots) indicates proximity between CD93 and MMRN2. F-actin was stained by phalloidin (red) and nuclei by Hoechst (blue). Scale bar: 20 mum. ( C ) Quantification of CD93 and MMRN2 interaction based on the number of positive signals per cell ( n = 3 independent experiments). Nonspecific signals (PLA negative controls) were also examined. ** P < 0.01; 1-way ANOVA with Dunnett's multiple-comparisons test. ( D - F ) Immunofluorescent staining of MMRN2, CD93, and CD31 in human grade IV glioma vessels ( D ), in orthotopic GL261 glioma vasculature ( E ), and in nontumor brain vasculature adjacent to a GL261 tumor ( F ). Scale bars in all pictures: 20 mum. ( G ) MMRN2 quantification in tumor and nontumor vessels of WT ( n = 3) and CD93 -/- ( n = 3) mice. Values represent mean +- SEM expressed as arbitrary units (AU) of MMRN2-positive area normalized by CD31-positive area. ** P < 0.01; 2-tailed t test.

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Figure 10 CD93 deficiency results in diminished beta 1 integrin activation and fibronectin deposition in GL261 vasculature. ( A ) Immunofluorescent staining of the active conformation of beta 1 integrin (9EG7) in GL261 tumor vasculature of WT and CD93 -/- mice. CD31 was used as vascular marker (red) and Hoechst as nuclear marker (blue). High-magnification images show the levels of 9EG7 staining associated with the vasculature in WT and CD93 -/- mice. Scale bars: 20 mum. ( B ) Immunofluorescent staining of fibronectin and CD31 in GL261 tumor vasculature of WT and CD93 -/- mice. High-magnification images show fibronectin associated with the vasculature in WT and CD93 -/- mice. Scale bars: 20 mum. ( C ) Tile scan of GL261 tumor in WT and CD93 -/- mice stained for fibronectin. Scale bars: 500 mum. ( D ) Quantification of fibronectin-positive signal in WT ( n = 4) and CD93 -/- ( n = 6) mice. Values represent mean +- SEM expressed as arbitrary units (AU) of fibronectin-positive area normalized by total tumor area. *** P < 0.0001; 2-tailed t test. ( E ) Quantification of tumor area in WT ( n = 4) and CD93 -/- ( n = 6) mice. Values represent mean +- SEM expressed as AU of the total tumor area determined by nuclei staining. * P < 0.05; 2-tailed t test. ( F ) 3D volume reconstruction of fibronectin (green) and CD31 (red) immunofluorescent staining in a GL261 vibratome section. Arrowheads indicate fibronectin fibers associated with vessels (CD31 positive). Scale bar: 20 mum.

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Figure 5 CD93 and MMRN2 colocalize in the tip cells during sprouting angiogenesis and are required for endothelial sprouting. ( A ) Immunofluorescent staining of CD93 and MMRN2 in the sprouting front (indicated by dotted line) of vessels formed in embryoid bodies (EBs) cultured in 2D on glass slides upon VEGF treatment. Differentiated endothelial cells were visualized by CD31 staining. Scale bars: 20 mum. Specific localization of CD93 and MMRN2 in CD31-positive sprouts and filopodia is shown in high-magnification images. ( B ) Immunofluorescent staining of CD93 and MMRN2 in EBs cultured in a 3D collagen gel. Scale bars: 200 mum. High-magnification images show a specific localization of CD93 in the tip cells and in filopodia. MMRN2 staining indicates its colocalization with CD31-positive sprouts and with CD93 in the tip cells. ( C ) CD31 immunofluorescent staining of shCD93- and shMMRN2-transfected EBs cultured in 2D glass slide. Untransfected (Ctrl) or empty vector-transfected (shCtrl) EBs were used as controls. Scale bars: 500 mum. ( D ) Quantification of CD31 sprouting expansion. Values represent mean +- SEM (at least n = 5 EBs per condition). *** P < 0.001; 1-way ANOVA with Dunnett's multiple-comparisons test. ( E ) CD31 immunofluorescent staining of shCD93- and shMMRN2-transfected or control EBs (Ctrl and shCtrl) cultured in 3D collagen gel. Scale bars: 500 mum. ( F ) Quantification of CD31-positive sprouts. Values represent mean +- SEM (at least n = 10 EBs per condition)

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Figure S1 Representative images of nestin ( A ) and Tmsb4x ( B ) staining in the mouse adult wild-type glomerulus visualized by fluorescent microscopy. ( C ) Merged images showing Tmsb4x (red) and nestin (green) staining; areas of colocalization are indicated by arrows. Representative images of Tmsb4x ( D ) and Cd31 ( E ) staining in the mouse adult wild-type glomerulus visualized by fluorescent microscopy. ( F ) Merged images showing Tmsb4x (green) and Cd31 (red) staining. Bar = 20 mum.

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FIGURE 3 Igfbp7/IGFBP7 is upregulated in the vasculature in response TBI. (A-C) The expression level of Igfbp7 in the TBI and control groups from the three data sets. From left to right, whole cortex, FACS sorted EC RNAseq, and scRNAseq data sets. **** indicates p < 0.0001. (D) The expression level of Igfbp7 in the main cell types from the scRNAseq study. AC, Astrocyte; EC, endothelial cell; EP, Ependymocyte; MG, Microglia; NE, Neuron; OL, Oligodendrocyte; OPC, Oligodendrocyte progenitor cell. (E) Immunofluorescence co-staining of CD31 (green) and IGFBP7 (red) in human samples. The TBI and control group were both operated from the right frontal. Scale bar, 50 mum.

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Fig. 2 MLKL regulates adhesion molecules expression as well as EC-leukocyte interaction. a Heatmap of normalized expression dynamics of top 2000 DEGs in vehicle and TNFalpha, NSA, or TNFalpha + NSA treated cells. K-means clustering analysis identified three clusters of DEG according to their expression patterns. Left panel shows the gene ontology biological process analysis of the three DEG clusters ( n = 3 biological triplicates). b , c Control or MLKL knockout HUVEC were stimulated with TNFalpha (50 ng/ml) for 6 h. Expression levels of adhesion molecules were determined by real-time PCR ( b ) and immunoblot ( c ). d , e Control or MLKL knockout HUVEC monolayers were pretreated with TNFalpha (100 ng/ml) for 12 h, then incubated with calcein-AM-labeled iBMDM for 30 min and adherent iBMDM were imaged ( d ) and quantitated ( e ). Calcein-AM, green. Scale bar, 100 mum. f , g Mlkl +/+ ( n = 3-4) and Mlkl -/ - ( n = 3-6) mice were intradermally injected with 100 ng TNFalpha (in 50 mul saline) into the right side of abdominal skin to induce local inflammation, with the same volume of saline injected into the left side as control. Four hours later, the leukocytes (rhodamine-6G + ) that adhered to the vessel wall were imaged by intravital confocal microscopy ( f ) and quantitated ( g ). Vessel were visualized with FITC-Dextran. h , i Eight hours after TNFalpha injection, the extravasated leukocytes were imaged ( h ) and quantitated ( i ). Leukocytes were stained with CD45 antibody an

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Figure S1 Localization of CD4 T Cells in the Healthy Mouse Brain, Related to Figure 1 (A) Perfused brain was stained for CD4 (green), laminin 4 (red), laminin alpha1 (white) and DAPI (blue), displaying individual channels and composite image. Representative image of CD4 T cell crossing laminin 4 barrier or (B) laminin alpha1 barrier within midbrain meningeal folds. (C) Representative image of a CD4 T cell undergoing transvascular movement in the hindbrain. (D) Representative images of CD4 T cells beyond the laminin 4/alpha1 barrier in the cerebellum, (E) hindbrain or (F) olfactory bulb. (G) Surface rendering of confocal images showing CD4 T cells (green), CD31 + vasculature (red), GFAP + astrocytes (magenta) and DAPI (blue). Representative images of CD4 T cells enclosed by the glia limitans in the mid-brain, and beyond the glia limitans in (H) the midbrain and (I) the cerebellum. (J) Perfused brain was stained for CD4 (green), CD31 (red), Iba1 (yellow) and DAPI (blue), displaying individual channels and composite image. Representative images of CD4 T cells in close proximity to microglia in the midbrain (K) or hindbrain. Scale bar = 20mum. (L) Representative confocal images showing CD4 T cells, immunostained using CD4 (green) and Foxp3 (red) staining located in mouse brain distal to the vasculature, adjacent to the vasculature, and in the intravascular space. Fluorescent-labeled lectin was used to label vasculature (white) and cell nuclei were stained with DAPI (blue). Scale

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FIG. 4 Morphological analysis of HSCs in liver fibrosis. (A) The liver sections of sham-operated, CCL 4 -treated, or bile duct-ligated Gucy1a1-EGFP mice were stained with collagen-1 and CD31 antibodies. The morphology of EGFP+ HSCs were skeletonized; cell bodies are in blue and cellular processes are in red. (B) Morphological analysis of EGFP+ HSCs in the liver sections of sham-operated, CCL 4 -treated, or bile duct-ligated Gucy1a1-EGFP mice. Statistical analyses were performed using one-way ANOVA with Tukey HSD test. ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. (C) Liver sections of CCL 4 -treated Gucy1a1-EGFP mice were stained with collagen1 and CD31. Stage-1, stage-2, and stage-3 HSCs were defined based on their relative distance to the collagen-1-positive stage-3 aHSCs. Zoomed-in image showing the morphology of qHSCs and aHSCs at stage 1, stage 2, and stage 3. Arrow indicate the skeletonized morphology of individual aHSCs. (D) Morphological comparison of qHSCs and HSCs at stage 1, stage 2, and stage 3. Statistical analyses were performed using one-way ANOVA with Tukey HSD test. ns: not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.

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The IL33-containing BAC in transgenic mice encodes human-specific regulatory patterns and demonstrates the importance of the 5 kb noncoding segment for proper IL33 expression. a Schematic of human BAC clone RP11-725F15 (166 kb) spanning the entire coding region of IL33 and its upstream region including the 20 kb asthma-associated interval and the 5 kb region of interest shaded in blue (black bars). To produce a human IL33 reporter strain, a cassette containing E2-Crimson with a stop sequence was inserted into exon 2, in frame with the IL33 translational start site (red dotted line). Transgenic mice were generated with either the full BAC (h IL33 Crim BAC) or a BAC containing a deletion of the 5 kb interval within the LD block (h IL 33 Crim BAC5kdel). b , c Immunofluorescence staining of mouse peripheral lymph node sections (left panels) and trachea tissue sections (right panels) of E2-Crimson in h IL33 Crim BAC mice ( b ) or h IL33 Crim BAC5kdel ( c ). Representative founder BAC transgenic lines are shown. Sections were stained with anti-E2-Crimson (red) and the mouse endothelial cell marker CD31 (green). Hoechst 33342 staining for nuclei is in blue. Pie charts show the distribution of Crimson expression (Crm) in each ""humanized"" BAC mouse line. d qPCR analysis of E2-Crimson mRNA obtained from lymph node, heart, and lung from both BAC strains is shown. Violin plot shows average dCT values (Crimson/Ppia) obtained from animals containing either the full BAC or the 5 kb deleti

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Figure 2. Myeloid-specific NG2 ablation impairs macrophage recruitment to tumors. Double immunostaining for CD11b (blue) and CD31 (green) in tumor sections from control ( A-C ) and Mac-NG2ko mice ( D-F ). Areas of overlap in z-stacks appear as pale blue ( C,F ). Quantification of macrophage abundance in tumors using the markers F4/80 ( G, I ) and CD11b ( H, J ). Data are plotted as the percentage of total tumor area occupied by marker pixels in 10-d tumors ( G, H ) and 16-day tumors ( I, J ). Scale bars = 20 mum. * p < 0.01 compared to controls.

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Figure 3. NG2-dependent recruitment of bone marrow-derived macrophages. Tumor sections from mice transplanted with EGFP-positive bone marrow from wild type ( A-C ) or germline NG2 donors ( D-F ) were used to localize bone marrow-derived macrophages in relation to CD31-positive blood vessels (red). Panels C and F are merged images of confocal z-stacks. ( G ) Quantification of EGFP pixels as a percentage of tumor area. In parallel, sections were immunostained for CD11b (red) and for PDGFRbeta (not shown) to identify EGFP-positive macrophages and pericytes, respectively, in mice transplanted with EGFP-positive bone marrow from wild type ( J-L ) or germline NG2 donors ( M-O ). Merged images of confocal z-stacks are shown in panels L and O. ( H ) Quantification of the percentage of EGFP-positive cells that are CD11b-positive. ( I ) Quantification of the percentage of EGFP-positive cells that are PDGFRbeta-positive. Scale bars = 20 mum. * p < 0.01 compared to controls.

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Figure 6. Loss of vascular N-cadherin expression following myeloid-specific ablation of NG2. Double immunostaining for CD31 (red) and N-cadherin (green) was used to assess endothelial cell expression of N-cadherin in control ( A-C ) and Mac-NG2ko tumor vessels ( D-F ). ( G ) Quantification of N-cadherin/CD31 colocalization as a percentage of total CD31. Double immunostaining for PDGFGRbeta (red) and N-cadherin (green) was used to evaluate pericyte expression of N-cadherin in control ( H-J ) and Mac-NG2ko tumor vessels ( K-M ). ( N ) Quantification of N-cadherin/PDGFRbeta colocalization as a percentage of total PDGFRbeta. Scale bars = 20 mum. * p < 0.01 compared to controls.

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Figure 7. Altered expression of VEGF-A following myeloid-specific ablation of NG2. Double immunostaining for VEGF-A (red) and CD31 (green) was used to quantify and localize VEGF-A expression in tumors from control ( A-C ) and Mac-NG2ko tumors ( D-F ). Overall expression of VEGF-A increases 3-fold in Mac-NG2ko tumors ( G ), consistent with the increased HIF-1alpha expression seen in Fig. 5I . However, VEGF-A in control tumors is highly localized to blood vessels, while vascular VEGF-A in Mac-NG2ko tumors is reduced 3-fold ( H ). Instead, non-vascular VEGF-A in Mac-NG2ko tumors is increased by a factor of 5 ( I ). Scale bars = 60 mum. * p < 0.01 compared to controls.

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Figure 4 Hematopoietic cell Vegfr2 inactivation, targeted with Vav-Cre , minimally impacts LLC tumor growth. ( a ) LLC tumor volume 20 days post tumor cell inoculation showing a delayed minimal decrease in tumor volume in Vav-Cre; Vegfr2 f/f versus Vegfr2 f/f control mice (designated Ctl) (n = 6). ( b , c ) Immunofluorescent staining ( b ) and quantitation ( C ) of CD31-positive tumor vasculature showing no impairment in tumor angiogenesis Vav-Cre; Vegfr2 f/f versus control mouse (n = 5). Two-way repeated-measures ANOVA with Sidak's multiple comparism-test (a) and two-tailed Student's t-test (c) were used to analyze significance (**p < 0.01) compared to control. ns, not significant. Scale bar, 100 mum.

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2 Stages of pre- and postnatal development of tracheal vasculature. A-L: Confocal micrographs of tracheal whole-mounts with blood vessels stained for PECAM-1 at E16.5 (A,E,I), at E17.5 (B,F,J), P2 (C,G,K) and P5 (D,H,L). A,B: The primitive vascular plexus of undifferentiated blood vessels is highly anastomotic at embryonic day (E) 16.5 (A) and E17.5 (B). C: Vascular pruning stage: Segmentation of vasculature at postnatal day (P) 2 by regression of the primitive plexus over cartilage rings (*). D: Vascular regrowth and adult stage: Adult vascular pattern starts to become evident at P5, after growth of horizontal capillaries over cartilage rings, where almost no vessels are present at P2. Vertically oriented arterioles and venules are present between the rings. E-H: Panels A-D shown in inverted polarity grayscale to emphasize the conspicuous differences in the vasculature patterns. I-L: Higher magnification views of the vascular architecture at E16.5, E17.5, P2, and P5. Scale bar = 80 mum in A-C,E-G, 100 mum in D,H, 20 mum in I-L.

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5 Pericyte coverage of tracheal vasculature. A-E: Confocal micrographs of tracheal whole-mounts stained for PECAM-1 (green) and desmin (red) comparing pericyte coverage of vasculature in mice at embryonic day (E) 17.5, postnatal day (P) 3, and P7. A: At E17.5 pericytes are sparse. B,C: During capillary regrowth, new vessels over cartilage rings have more complete pericyte coverage at P3 and P7 than at E17.5. Scale bar = 20 mum. D,E: Measurements of pericyte maturity expressed as (D) percentage of tracheal capillary length accompanied by pericytes and (E) number of pericyte cell bodies per millimeter of capillary. Asterisks indicate significantly fewer pericytes ( P < 0.05, Dunnett test) than in adult mice (P70).

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6 Distribution of activated caspase-3 and phosphohistone H3 at postnatal day (P) 3/P7. A-C: Whole-mount of P3 trachea stained for activated caspase-3 immunoreactivity to mark apoptotic cells and PECAM-1 to mark endothelial cells. Some activated caspase-3-positive cells (arrows) are associated with blood vessels. B and C are enlargements of boxes in A and B. D-F: Whole-mount of P7 trachea stained for phosphohistone H3 immunoreactivity to mark dividing cells and for PECAM-1. Many cells stain for phosphohistone H3 (arrows). Although most (*) are not associated with blood vessels, some were in the wall or lumen of blood vessels. E and F are enlargements of boxes in D and E. Scale bar in D = 140 mum in A,D, 50 mum in B,E, 20 mum in C,F.

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7 Hypoxia in newborn mouse trachea. A-H: Whole-mounts of mouse tracheas at postnatal day (P) 0 or P3 stained for hypoxic cells (pimonidazole, green) and blood vessels (PECAM-1, red). A-C: At P0, pimonidazole staining of tracheal epithelium is strong and, like the vascular plexus, relatively uniformly distributed. D-F: However, at P3 pimonidazole staining is weaker in regions between cartilage rings and is absent in regions over cartilages where blood vessels are also absent except in scattered hypoxic regions (*). G,H: Enlargements of regions in boxes in C and F. I,J: Cross-section of P0 trachea showing the epithelium is stained for pimonidazole (I) and HIF-1alpha (J), but cartilages (*) are not. K: Negative control shows little staining by secondary antibodies when pimonidazole and HIF-1alpha primary antibody were omitted. Scale bar in K = 140 mum in A-F,I-K, 50 mum in G,H.