Journal: Cell Reports Medicine

Article Title: A PD-1-targeted, receptor-masked IL-2 immunocytokine that engages IL-2Rα strengthens T cell-mediated anti-tumor therapies

doi: 10.1016/j.xcrm.2024.101747

Figure Lengend Snippet:

Article Snippet: CD45 (TIL) MicroBeads, mouse , Miltenyi Biotec , Cat# 130-110-618.

Techniques: Purification, Blocking Assay, Control, Recombinant, Staining, Luciferase, Isolation, Cell Isolation, Biomarker Discovery, Amplification, Knock-In, Plasmid Preparation, Expressing, Software

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) Representative flow cytometry graphs of CD11b and GFP in peripheral blood of KDRGFP mice bearing gliomas. Astrocytic tumors were generated by RCAS/TVA-mediated overexpression of PDGF as described in Methods. Low-grade gliomas were observed by week 5 and high grade by weeks 7–8. Controls (CTL) were mice bearing an intact Kdr locus without GFP knockin. (B) Quantification of CD11b+KDRGFP+ cell frequency in peripheral blood of RCAS/TVA tumor mice at low-grade and high-grade stages. ***P < 0.001, Student’s t test. LEU, Leukocytes. (C) Further characterization of murine CD11b+KDRGFP+ cells in peripheral blood by Ly6C and Ly6G staining. (D) Quantification of Ly6G+ and Ly6C+ frequency out of total CD11b+ cells in the CD11b+KDRGFP+ and CD11b+KDRGFP– populations in peripheral blood at high-grade stage. **P < 0.01, for Ly6Chi cells, Student’s t test. (E) CFU assays were performed on Lin–KDRGFP+ hematopoietic cells from BM, and macrophage (M) colonies and macrophage/granulocyte (GM) colonies, but not granulocyte (G) colonies, were observed. Quantification of various colony types formed from lineage-negative KDRGFP– or KDRGFP+ cells. ***P < 0.001, in granulocytes, Student’s t test. (F) Gene expression (quantitative RT-PCR) of various markers, including Kdr in in vitro–cultured BM Lin– cells exposed to GL261-conditioned medium at different time points. *P < 0.05; **P < 0.01; ***P < 0.001, 1-way ANOVA. Data are shown as mean ± SD.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Flow Cytometry, Generated, Over Expression, Knock-In, Staining, Gene Expression, Quantitative RT-PCR, In Vitro, Cell Culture

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: Lethal dose–irradiated C57BL/6 mice were transplanted with Ubc-GFP Rosa26-CreERT2Kdrfl/fl and Rosa26-CreERT2KDRfl/+ BM cells, and GL261 tumors were implanted after BM engraftment. (A) Peripheral white blood cells were analyzed on side scatter (SSC) and GFP by flow cytometry. The GFP+ and GFP– populations were gated for further analysis. CD11b versus GFP (B), Ly6C versus Ly6G (C), and CD3 versus B220 (D) are shown on both GFP+ and GFP– populations. (E) BM cells were also analyzed on side scatter and GFP. (F) Lineage-negative cells were gated for analysis of HSCs and HPCs by c-Kit versus Sca-1 (G). (H) HPCs (Lin–c-Kit+Sca-1–) were further characterized by FcγR versus CD34 within both GFP+ and GFP– populations. The experiment was conducted 5 times.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Irradiation, Flow Cytometry

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) The unsupervised principal component analysis of significantly altered genes in Lin–c-Kit+Sca-1– from naive mice, Lin–c-Kit+Sca-1– KDR-GFP+ cells, and Lin–c-Kit+Sca-1–KDR-GFP– cells from BM of tumor-bearing mice. (B) Lin–c-Kit+Sca-1– cells from naive mice (black), Lin–c-Kit+Sca-1– KDR-GFP+ cells (red), and Lin–c-Kit+Sca-1–KDR-GFP– cells (green) from BM of tumor-bearing mice, based on similarity of gene profiles. PC1, principal component 1. (C) The candidate genes (P < 0.05, >1.5-fold change; Lin–c-Kit+Sca-1– KDR-GFP+ versus Lin–c-Kit+Sca-1–KDR-GFP–) were divided according to the subset with the highest expression and analyzed for categories with significant enrichment (P < 0.05) of categories in GO biologic processes using DAVID tools. Similar categories were grouped accordingly. The presence of association between functions and genes was color highlighted (black [negative] versus green [positive]). (D) Expression of Id2 in various lineages of hematopoietic cells. ***P < 0.001, 1-way ANOVA. (E) Expression of ID2 in HPCs (CD45–CD34+) from patients with low-grade or high-grade gliomas. ***P < 0.001, 1-way ANOVA. n = 21. (F) Expression of ID2 in CD11b+ blood cells from patients with low-grade or high-grade gliomas. ***P < 0.001, 1-way ANOVA. n = 20. Data are shown as mean ± SD.

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Expressing

Journal: The Journal of Clinical Investigation

Article Title: A proangiogenic signaling axis in myeloid cells promotes malignant progression of glioma

doi: 10.1172/JCI86443

Figure Lengend Snippet: (A) Chimeric C57BL/6 mice transplanted with Id2–/– BM cells (Id2+/+ BM cells as control) were implanted with luciferase-labeled GL261 tumors intracranially. Tamoxifen was applied at day 3 after implantation. The tumors were monitored by bioluminescence. Representative images were taken at day 14. The color bar on the right represents photon intensity. The experiments had 2 replicates. The tumor growth curve is based on bioluminescence. n = 10. **P < 0.01, 1-way ANOVA. (B) Flow cytometry analysis of peripheral blood cells on CD11b, Ly6C, and Ly6G in Id2+/+ BMT and Id2–/– BMT groups. Quantification of CD11b+ cell frequency out of total white blood cells and of Ly6Chi cell frequency out of CD11b+ cells. **P < 0.01, 1-way ANOVA, for each group. n = 7. (C) Tubule formations of HCMEC/D3 (GFP) cocultured with lineage-negative Id2+/+, Id2–/–, Id2–/– scrambled sequence (SC) control, or Id2–/– KDR overexpression (OE) HPCs pretreated with TGF-β/GM-CSF. Quantifications of tubule lengths in the indicated group. ***P < 0.0001, 1-way ANOVA. n = 6. (D) Growth factor–reduced Matrigel plugs of lineage-negative Id2+/+, Id2–/–, Id2–/– SC CTL, or Id2–/– KDR overexpression HPCs pretreated with TGF-β/GM-CSF. Blood vessels (red) were perfused with rhodamine-dextran. Lower panels show magnified views to highlight vascular permeability. Quantifications of based blood vessels density (BVD) in each group. **P < 0.01, 1-way ANOVA. n = 6. Data are represented as mean ± SD. Scale bars: 50 μm (D, upper panels); 20 μm (C, D, lower panels).

Article Snippet: Uncommitted BM progenitors, highly enriched in HSCs, were negatively selected using either the Lineage Cell Depletion Kit for mouse (Miltenyi Biotec) or the Hematopoietic Progenitor Enrichment Kit (StemCell Technologies) following the manufacturer’s protocols.

Techniques: Control, Luciferase, Labeling, Flow Cytometry, Sequencing, Over Expression, Permeability

Journal: Cell Reports Medicine

Article Title: A PD-1-targeted, receptor-masked IL-2 immunocytokine that engages IL-2Rα strengthens T cell-mediated anti-tumor therapies

doi: 10.1016/j.xcrm.2024.101747

Figure Lengend Snippet:

Article Snippet: Fragments were digested into single-cell suspensions using the Miltenyi tumor isolation kit (130-096-730).

Techniques: Purification, Blocking Assay, Control, Recombinant, Staining, Luciferase, Isolation, Cell Isolation, Biomarker Discovery, Amplification, Knock-In, Plasmid Preparation, Expressing, Software

Journal: Frontiers in Immunology

Article Title: The role of interleukin-18 and interleukin-18 binding protein in K/BxN serum transfer-induced arthritis

doi: 10.3389/fimmu.2023.1215364

Figure Lengend Snippet: Neutrophils express IL-18BP in K/BxN STA joints. (A) Determination of Il18 and Il18bp mRNA levels in the right ankle joints of naïve (black = 6) and K/BxN arthritic (blue = 10) littermates, after 7 days from the first K/BxN IgG injection, relative to Gapdh . Data are shown as the mean ± SEM of values. Statistical analysis was performed using a Kruskal–Wallis test. A significant p -value is represented * p ≤ 0.05. (B–E) Immunofluorescent staining of (B) circulating white blood cells and (C–E) left ankle joints of WT and IL-18BP-Tomato-KI mice after 7 days from the first PBS or K/BxN IgG injection. The sections are stained for DAPI (blue) and RFP (red) as well as for endothelial cells using anti-CD31 antibody (C) , synovial neutrophils using anti-Ly6G antibody (D) , and macrophages using anti-F4/80 antibody (E) (green). The scale is present on panels (A) 10 μm, (B) 20 μm, (C) 40 μm, and (D) 20 μm.

Article Snippet: Knock-in (KI) Il18bp tdTomato reporter mice were created by Ingenious Targeting Laboratory (Ronkonkoma, NY, USA), as described ( ).

Techniques: Injection, Staining

Journal: Frontiers in Immunology

Article Title: The role of interleukin-18 and interleukin-18 binding protein in K/BxN serum transfer-induced arthritis

doi: 10.3389/fimmu.2023.1215364

Figure Lengend Snippet: Clinical severity and inflammatory marker expression of K/BxN STA in WT and IL-18BP KO mice. WT (blue, n = 20) and IL-18BP KO (red, n = 20) littermates were injected i.p. with IgG purified from 150 μl of K/BxN serum on days 0 and 2. Clinical scores were blindly evaluated, daily up to day 7. (A) Mean arthritis severity, obtained from the sum of arthritis severity score of all 4 paws on a scale of 0 to 12. (B) Number of arthritic paws (arthritis severity score ≥1). (C) Incidence of arthritis, seen as percentage of mice with ≥1 joint with arthritis score ≥1. (D) Percentage of body weight variation (%). The results are shown as the mean ± SEM. (E) Determination of mRNA levels of Il18bp, Il18, Il1b, Il6, Il1r2, Ifng, Cxcl9, Cxcl1 , and Cxcl2 in ankle joints. Total RNA was isolated from right ankle joints of WT (blue, n = 10) and IL-18BP KO (red, n = 10) at day 7 after the first K/BxN IgG injection for qRT-PCR analysis. Results represent Il18bp, Il18, Il1r2, Il6, Ify, Il1b, Cxcl9, Cxcl1 , and Cxcl2 mRNA expression levels relative to Gapdh mRNA levels. Data are shown as the mean ± SEM of values. Statistical analysis was performed using a Mann–Whitney test. p -values not shown signify a lack of statistical significance.

Article Snippet: Knock-in (KI) Il18bp tdTomato reporter mice were created by Ingenious Targeting Laboratory (Ronkonkoma, NY, USA), as described ( ).

Techniques: Marker, Expressing, Injection, Purification, Isolation, Quantitative RT-PCR, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: The role of interleukin-18 and interleukin-18 binding protein in K/BxN serum transfer-induced arthritis

doi: 10.3389/fimmu.2023.1215364

Figure Lengend Snippet: K/BxN STA severity in WT and IL-18 KO littermates. WT (blue, n = 7) and IL-18 KO (orange, n = 7) littermates were injected i.p. with IgG purified from 150 μl of K/BxN serum on days 0 and 2. Clinical scores were blindly evaluated, daily up to day 7. (A) Mean arthritis severity, obtained from the sum of arthritis severity score of all 4 paws on a scale of 0 to 12. (B) Number of arthritic paws (arthritis severity score ≥1). (C) Incidence of arthritis seen as percentage of mice with ≥1 joint with arthritis score ≥1; WT incidence is identical to IL-18 KO) and (D) body weight variation (%). (E) Determination of mRNA levels of Il18bp, Il18, Il1b, Il6, Il1r2, Ifng, Cxcl9, Cxcl1 , and Cxcl2 in ankle joints. Total RNA was isolated from right ankle joints for qRT-PCR analysis. Results represent mRNA expression levels relative to Gapdh . Data are shown as the mean ± SEM of values. Statistical analysis was performed using a Mann–Whitney test. p -values not shown signify a lack of statistical significance. *** denotes p ≤ 0.001.

Article Snippet: Knock-in (KI) Il18bp tdTomato reporter mice were created by Ingenious Targeting Laboratory (Ronkonkoma, NY, USA), as described ( ).

Techniques: Injection, Purification, Isolation, Quantitative RT-PCR, Expressing, MANN-WHITNEY

Journal: The Journal of Clinical Investigation

Article Title: Collagen type VI regulates TGF- β bioavailability in skeletal muscle in mice

doi: 10.1172/JCI173354

Figure Lengend Snippet: ( A ) Double-homozygous Ltbp4 hom / Col6a2 –/– mice have weight similar to that of Col6a2 –/– mice and are smaller and lighter than WT and Ltbp4 het and Ltbp4 hom littermates. ( B ) H&E staining of tibialis anterior muscle of double-homozygous Ltbp4 hom /Col6a2 –/– mice shows dystrophic features similar to those in Col6a2 –/– mice (25 weeks old). Scale bars: 50 μm. ( C ) Although Ltbp4 hom / Col6a2 –/– mice have slightly higher forelimb grip strength compared with Col6a2 –/– mice, it remains markedly lower than that of WT Ltbp4 het and Ltbp4 hom littermates. ( D ) Physiological parameters in isolated EDL muscle of 25-week-old Ltbp4 hom / Col6a2 –/– mice were unchanged in comparison with Col6a2 –/– mice. For C and D , statistical comparisons were performed by 2-way ANOVA and Tukey’s adjustment for multiple comparisons. **** P < 0.0001. ( E ) After repeated eccentric contractions, tetanic force declined precipitously in isolated double-homozygous Ltbp4 hom / Col6a2 –/– and Col6a2 –/– EDL muscles, more prominently in male animals compared with female animals. Statistical analysis was performed using linear mixed models and Bonferroni adjustment for multiple comparisons. Error bars represent SEM.

Article Snippet: The Ltbp4 c/s knockin mice were generated by InGenious Targeting Laboratory as previously described ( ) with an Ltbp4 p.Cys1260Ser substitution and re-derived at the National Institute of Mental Health (NIMH) transgenic mouse core from mouse sperm straws provided by Daniel Rifkin’s laboratory at New York University.

Techniques: Staining, Isolation, Comparison, Muscles

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: In situ binding of recombinant integrins to frozen sections of mouse embryonic tissues. (A–C) Recombinant integrin α8β1 (green) bound to E15.5 dorsal skin (A), lung (B), and choroid plexus (C) cryosections. The top and bottom panels show fluorescence images for wild-type and Qbrick −/− littermates, respectively. (D–F) Integrins α3β1 (D), α6β4 (E), and α7X2β1 (F) bound to dorsal skin cryosections from wild-type (top) and Qbrick −/− (bottom) littermates were visualized (green). (G and H) Integrin α8β1 (green) bound to dorsal skin cryosections from Frem2 my /+ and Frem2 my / my (G) and Grip1 eb /+ and Grip1 eb / eb (H) mice. The top and bottom panels show fluorescence images for control heterozygotes and homozygotes, respectively. The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). Immunoreactivity against QBRICK (green) is also shown. Arrowheads indicate the location of BMs. The asterisks indicate nonspecific binding of recombinant integrins or the anti-QBRICK antibody to the cornified epithelium. Bars, 20 µm.

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: In Situ, Binding Assay, Recombinant, Fluorescence, Control

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: Impaired binding of integrin α8β1 in the developing kidney of Qbrick −/− mice. (A) Unilateral renal agenesis observed in Qbrick −/− mice. Bar, 5 mm. (B and C) The left panels show integrin α8β1 (green) bound to the BMs of the E10.5 mesonephric duct (B) and E11.5 ureteric bud (C) of wild-type (top) and Qbrick −/− (bottom) embryos. The right panels show the BMs counterstained with an anti–laminin-γ1 chain antibody (magenta). (D and E) QBRICK immunofluorescence (green) in the developing kidney of wild-type (top) and Qbrick −/− (bottom) embryos at E10.5 (D) and E11.5 (E). The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). The open and closed arrowheads indicate the mesonephric duct and ureteric bud, respectively. The asterisks indicate nonspecific binding of the anti-QBRICK antibody to the mesenchyme. Bars, 50 µm. (F) The expression levels of Gdnf and Pax2 transcripts in the E11.5 metanephros of Qbrick +/− control (open bars) and Qbrick −/− (shaded bars) littermates were determined by quantitative RT-PCR and normalized by the expression level of Gapdh . The expression level in control mice was set at 1. Each bar represents the mean ± SD (error bars; n = 3). **, P < 0.01, significant difference by Student’s t test.

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Binding Assay, Immunofluorescence, Expressing, Control, Quantitative RT-PCR

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: Generation of Qbrick RGE/RGE mice. (A) Schematic representation of the targeted mutation of Qbrick . Open boxes represent exons. The knock-in construct was designed to replace exon 4, which contains the codon encoding Asp207, with a mutated exon (open box with asterisk) in which the codon encoding Asp207 was substituted with a codon encoding Glu207. The probes used for Southern blotting are indicated by bold lines. N, NcoI restriction site; TK, thymidine kinase. (B) Direct DNA sequencing of the wild-type and Qbrick RGE/RGE genomes. The numbers indicate the positions of the amino acids in the primary sequence of QBRICK protein. (C) Southern blot analyses of genomic DNA from wild-type, Qbrick RGEneo/+ , and Qbrick RGE/+ offspring after digestion with NcoI. Fragments of 5.3 and 7.7 kbp are detected with probe 1 and probe 2, respectively, indicating that the expected homologous recombination occurred. The detection of a 6.1-kbp fragment with probe 2 indicates that the neomycin resistance gene has been removed from the Qbrick RGEneo allele by the Cre-loxP system. (D and E) Immunofluorescence staining (green) for QBRICK in E15.5 dorsal skin (D) and E11.5 ureteric buds (E). The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). (F–H) Qbrick RGE/RGE mice develop normally without any defects in the kidneys (asterisks in F), eyelids (G), and digits (H). (I and J) Binding of recombinant integrin α8β1 (green) to E15.5 dorsal skin (I) and E11.5 ureteric buds (J). The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). In panels D, E, I, and J, the top and bottom panels show fluorescence images for wild-type and Qbrick RGE/RGE littermates, respectively. The expression levels of QBRICK are comparable between wild-type and Qbrick RGE/RGE littermates. It should be noted that the binding of integrin α8β1 to the epidermal BMs of the E15.5 dorsal skin (I) as well as the E11.5 ureteric buds (J) is comparable between wild-type and Qbrick RGE/RGE littermates. Bars: (D and I) 20 µm; (E and J) 50 µm; (F) 5 mm.

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Mutagenesis, Knock-In, Construct, Southern Blot, DNA Sequencing, Sequencing, Homologous Recombination, Immunofluorescence, Staining, Binding Assay, Recombinant, Fluorescence, Expressing

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: Impaired expression of nephronectin and MAEG in FS model mice. (A–F) Immunofluorescence staining (green) for nephronectin (A, C, and E) and MAEG (B, D, and F) in the dorsal skin of Qbrick −/− (A and B), Frem2 my / my (C and D), and Grip1 eb / eb (E and F) mice, and their control wild-type or heterozygous littermates. (G–J) Immunofluorescence staining (green) for nephronectin (G and H) and MAEG (I and J) at the E10.5 mesonephric duct (G and I; open arrowheads) and E11.5 ureteric bud (H and J; closed arrowheads) in wild-type (top) and Qbrick −/− (bottom) littermates. The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). Bars: (A–F) 20 µm; (G–J) 50 µm. (K and L) Immunoblot detection of laminin β1 and γ1 chains (LN), integrin α8 (Intα8), nephronectin (Npnt), and MAEG in protein extracts from the kidney (K) and skin (L) of E15.5 Qbrick −/− mice and their wild-type littermates. (M–O) Immunoblot signal intensities of laminin, integrin α8, nephronectin, and MAEG in the kidney (left) and skin (right) of E15.5 wild-type (open bars) and Qbrick −/− (shaded bars; M), E15.5 Frem2 my /+ (open bars) and Frem2 my / my (shaded bars; N), and E17.5 Grip1 eb /+ (open bars) and Grip1 eb / eb (shaded bars; O) mice. The signal levels in control mice were set at 1. Each bar represents the mean ± SD (error bars; n = 3–6). *, P < 0.05; **, P < 0.01; ***, P < 0.001, significant differences by Student’s t tests. (P) Titration curves of recombinant integrin α8β1 bound to the GST-fused NV domain of QBRICK (open triangles), GST-fused RGD linker segment of nephronectin (open diamonds), GST-fused RGD linker segment of MAEG (open squares), and GST (closed circles). Each point represents the mean ± SEM ( n = 3).

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Expressing, Immunofluorescence, Staining, Control, Western Blot, Titration, Recombinant

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: The binding of integrin α8β1 to BMs is not compromised in Maeg ΔRGD mice. (A) Schematic views of MAEG and MAEG ΔRGD , a deletion mutant lacking the linker segment. The sites recognized by the anti-MAEG antibodies (Anti-linker and Anti-MAM) are also shown. (B) Schematic representation of the targeted mutation of Maeg . Open boxes represent exons. The targeting construct was designed to replace exons 9 and 10, which encode the linker region, with PGKneo. The probes used for Southern blotting are indicated by bold lines. B, BamHI restriction site; TK, thymidine kinase. (C) Southern blot analysis of genomic DNA from wild-type, Maeg ΔRGD/+ , and Maeg ΔRGD/Y mice digested with BamHI. The detection of a 6.3-kbp fragment instead of an 8.2-kbp fragment indicates the presence of the mutated allele. (D) Direct DNA sequencing of the Maeg ΔRGD/Y -derived RT-PCR product demonstrating that the open reading frame has not shifted. The product encodes a polypeptide lacking amino acids 258–391 of the 550–amino-acid full-length sequence. The boundary between exons 8 and 11 is shown by the red broken line. The numbers indicate the positions of the corresponding amino acids in the wild-type MAEG sequence. (E and F) In E15.5 dorsal skin epidermal BMs, MAEG is comparably detected by immunofluorescence (green) in both Maeg +/Y and Maeg ΔRGD/Y littermates with the anti-MAM antibody (E), but is only detected in Maeg +/Y embryos by the anti-linker antibody (F). (G, H, and K) Immunofluorescence staining for QBRICK (G, green) and nephronectin (H, green), and binding of recombinant integrin α8β1 (K, green) to epidermal BMs of the E15.5 dorsal skin are comparable between Maeg +/Y and Maeg ΔRGD/Y littermates. The BMs were counterstained with an anti–laminin-γ1 chain antibody (magenta). In each set of panels, the top and bottom panels show fluorescence images for wild-type Maeg +/Y and Maeg ΔRGD/Y littermates, respectively. Bars, 20 µm. (I and J) Immunoblot signal intensities of laminin β1 and γ1 chains (LN), integrin α8 (Intα8), and nephronectin (Npnt) in the kidney (I) and skin (J) of E15.5 control Maeg +/Y (open bars) and Maeg ΔRGD/Y (shaded bars) mice. The signal levels in control mice were set at 1. Each bar represents the mean ± SD (error bars; n = 3).

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Binding Assay, Mutagenesis, Construct, Southern Blot, DNA Sequencing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Sequencing, Immunofluorescence, Staining, Recombinant, Fluorescence, Western Blot, Control

Journal: The Journal of Cell Biology

Article Title: Basement membrane assembly of the integrin α8β1 ligand nephronectin requires Fraser syndrome–associated proteins

doi: 10.1083/jcb.201203065

Figure Lengend Snippet: A model for renal defects in FS. In the developing metanephros, QBRICK and other FS-associated proteins associate with nephronectin in the sublamina densa region and secure its localization at BMs. Metanephric mesenchymal cells interact with nephronectin through integrin α8β1. Stimulation of integrin α8β1 induces Gdnf , which is necessary for further development of the metanephros (left). In FS, nephronectin is not stably deposited at BMs, owing to the loss of FS-associated proteins, and is eventually degraded. The loss of nephronectin leads to failure of Gdnf induction and subsequent metanephric development (right).

Article Snippet: The primary antibodies used were: rabbit anti-laminin (L9393; Sigma-Aldrich), goat anti–mouse integrin α8 (R&D Systems), rabbit anti-nephronectin , and rabbit anti-MAEG ( ).

Techniques: Stable Transfection

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: 1A. To determine which cell type binds mCRP in the brain after the intraperitoneal (i.p.). injection, double immunostaining of mCRP and different types of cell markers was conducted in the cortex and hippocampal DG region. Cell markers, including the neuronal marker NeuN, the microglial marker Iba1, the astrocyte marker GFAP, vascular marker Lectin and the endothelial cell marker CD31 and CD144, were applied individually. Correlation coefficients and R values were calculated for each protein pair detected and are shown. 1B Immunofluorescence analysis of isolated brain microvessels from mice treated with mCRP (purple) were stained with antibodies for endothelial cell components CD144 (red), vWF (green), CD31 (red) and its phosphorylation pCD31 (green). Nuclear stained with DAPI. The bar is 20 µm. 1C. Representative images of cortex stained with CD31 (red) and mCRP (green) and merged (yellow) are shown to observe mCRP deposits in CD31 positive regions. The yellow fluorescence intensities were quantified and compared among different genotype mice with i.p. mCRP treatment. p = 0.0016, n = 11-14 mice in each group. 1D. Representative images of double immunostaining of phos-CD31 (pCD31, green) and CD31 (red) and the merged images (yellow), nuclei stained with DAPI, on the cortex of WT mice, ApoE -/- mice and mice expressing different ApoE genotypes after i.p. treatment with PBS vs. mCRP are shown. Total CD31 and pCD31 were quantified by fluorescence intensity and the pCD31/CD31 ratio (ApoE4 p < 0.001, ApoE -/- p < 0.001) total CD31 (WT p = 0.03, ApoE3 p = 0.03, ApoE4 p = 0.001), n = 7-19 in each group. 1E. Western blots showed that mCRP increased the level of pCD31 and decreased the CD31 expression levels in the hippocampal region in ApoE4 mice but not in ApoE3 or ApoE2 mice. 1F. Proximity ligation assay (PLA) was performed on the cortex (upper panel) and hippocampal CA3 region (lower panel) to examine the binding of mCRP and CD31 in WT mice and mice expressing different ApoE genotypes after i.p. injection of PBS vs. mCRP. Positive PLA fluorescence signals are shown in orange; nuclei were stained with DAPI (blue). Quantifications of orange fluorescence were conducted and are shown: WT vs. ApoE4 p < 0.0001, ApoE2 vs. ApoE4 p < 0.0001, and ApoE3 vs. ApoE4 p = 0.0002 for cortex; ApoE2 vs. ApoE4 p = 0.0004 and ApoE3 vs. ApoE4 p = 0.0018 for CA3. n = 8 in each group. 1G. Representative images of primary CD31 + BECs from WT mice treated with mCRP in vitro at day 5 are shown. PLA (orange) was performed to detect colocalization/binding between mCRP (purple) and CD31 (green). pCD31 (red) was detected by a specific antibody, and the nuclei were stained with DAPI. The scale bar is 10 µm. 1H. To determine whether the direct effects of mCRP on endothelia are dose-dependent and/or time-dependent, primary CD31 + BECs were treated with various concentrations of mCRP for 1, 4, 8 and 24 h. Quantification is shown of the mCRP deposits on the surface of cells (upper left panel), the binding affinity of mCRP with CD31 (lower left panel), the CD31 expression level (upper right panel) and the ratio of pCD31 normalized against total CD31 (lower right panel) under different concentrations of mCRP and time course. mCRP (10 µg/ml) showed maximum binding to CD31 after 8 hours of incubation ( p = 0.0002). mCRP decreased the expression of CD31 within the first hour of incubation ( p = 0.04) and increased the levels of pCD31 after incubation for up to 24 hours ( p < 0.001) in a dose-dependent manner. At least three independent experiments were conducted for each condition. 1I. Primary BECs were transfected with CD31-targeting siRNA to effectively knockdown CD31 expression ( p < 0.001) before adding mCRP (10 µg/mL) stimulation. Fluorescence immunostainings with antibodies, mCRP (purple), CD31 (green) and pCD31 (red) were shown. Nuclear was stained with DAPI. The quantifications were shown that compared to control cells, BECs after silencing CD31 with siRNA had lower mCRP binding ( p = 0.04). 1J. Primary BECs were incubated with CD31-specific antibody followed by adding mCRP (10 µg/mL) stimulation. A significant decreased pCD31 levels were observed in BECs after blocking CD31 by the antibodies ( p < 0.001). Data are shown as the mean ± SEM. One or two -way ANOVA with Tukey’s post hoc test and Pearson or Spearman correlation test were applied. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. The scale bar is 50 µm.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Injection, Double Immunostaining, Marker, Immunofluorescence, Isolation, Staining, Phospho-proteomics, Fluorescence, Expressing, Western Blot, Proximity Ligation Assay, Binding Assay, In Vitro, Incubation, Transfection, Knockdown, Control, Blocking Assay

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: 2A. Representative images of double immunostaining of ApoE (green) and CD31 (red) and the merged images (yellow) of the cortex sections of WT vs. different ApoE knock-in mice are shown. The ApoE + fluorescence intensity was compared between PBS and mCRP-treated mice and reached a significant difference only in the ApoE2 mice ( p = 0.02). n = 11-14 mice in each group. 2B. PLA was applied to detect the binding of ApoE and CD31 on cortex sections from different ApoE genotype mice treated with PBS vs. mCRP. Positive PLA fluorescence signals are shown in orange, and nuclei are stained with DAPI (blue). Quantifications of orange fluorescence intensity of ApoE-CD31 binding in the cortex are shown and reached a significant difference only in ApoE2 mice ( p = 0.04). PLA (mCRP-CD31) levels were negatively correlated with PLA (ApoE-CD31) in the brain (r = -0.51, p = 0.005). n = 8 in each group. 2C. Representative images of primary CD31 + BECs treated with 0.3 µM ApoE2, ApoE3 or ApoE4 protein are shown. The interaction/binding between ApoE (purple) and CD31 (green) was detected by PLA (orange). The positive PLA signal and CD31 protein level were quantified and compared. ApoE4 protein had the lowest binding with CD31 compared to ApoE2 ( p = 0.0002) and ApoE3 ( p = 0.0013) proteins. In addition, ApoE4 exhibited the most significant decrease in CD31 expression in CD31 + BECs compared to ApoE2 ( p < 0.001) and ApoE3 ( p = 0.04). The scale bar is 50 µm. 2D. To examine how different ApoE isoform proteins influence the effect of mCRP on endothelia, primary CD31 + BECs were preincubated with different concentrations of ApoE2, ApoE3 or ApoE4 protein for one hour followed by the addition of 10 µg/mL mCRP for 6 hours. Quantification of the CD31 expression level (upper left panel), the binding of ApoE-CD31 (upper right panel) and mCRP-CD31 (lower left panel) and the pCD31/CD31 ratio (lower right panel) were conducted. *Three ApoE isoform proteins were compared for each concentration, and the statistical significance is shown. #The comparison among different doses of protein for one isoform of ApoE; at least three independent experiments were conducted. Data are shown as the mean ± SEM. One or two -way ANOVA with Tukey’s post hoc test and Pearson correlation test were applied. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Double Immunostaining, Knock-In, Fluorescence, Binding Assay, Staining, Expressing, Concentration Assay, Comparison

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: 3A. After the i.p. treatment of PBS (top column) vs. mCRP (bottom columns), 3D images of cortex sections stained for CD31 (red) in WT, different ApoE-expressing and ApoE -/- mice were used to examine cerebrovasculature. The length of CD31-positive microvasculature in the cortex was quantified and compared between the PBS and mCRP groups in WT ( p = 0.01), ApoE3 ( p < 0.0001), ApoE4 ( p < 0.0001) and ApoE -/- ( p = 0.04) mice. # Comparison of ApoE genotypes in the PBS group, ApoE4 vs WT p = 0.04, ApoE2 p < 0.0001, ApoE3 p < 0.0001. n = 7-19 mice in each group. 3B. To examine the impact of elevated peripheral mCRP on cerebrovascular damage, we conducted immunostaining of a vascular damage biomarker, von Willebrand factor (vWF, green), and CD31 (red), in the cortex of WT vs. different ApoE knock-in mice. Representative images of each genotype treated with PBS vehicle (left columns) and mCRP (right columns) are shown. The numbers of vWF-positive endothelial cells in the cortex were compared among different genotype mice in the absence and presence of i.p. mCRP and showed statistical significance only in the ApoE4 group ( p = 0.04). A negative correlation between the length of CD31 + microvessels and the expression of vWF in the cortex was shown (r = -0.38, p = 0.0006). n = 5-6 in each condition. 3C. Western blots of different inflammatory and vascular-related proteins, including phosphorylated eNOS (p-eNOS), eNOS, NF-κB and CD144, in the cortex were conducted to examine the effects of mCRP on these proteins in each ApoE genotype. Peripheral mCRP significantly increased the expression of p-eNOS ( p = 0.04) and NF-κB ( p = 0.006) only in ApoE4 mice. 3D. Primary BECs were transfected with CD31-targeting siRNA to effectively knockdown CD31 expression ( p < 0.001) before adding mCRP (10 µg/mL) stimulation. Representative images of primary BECs silencing of CD31 with mCRP treatment were stained with antibodies vWF(green), NF-κB (red), p-eNOS (green). The nuclei was stained with DAPI. The quantifications were shown that lower levels of vWF ( p < 0.001), NF-κB ( p = 0.003) but higher level of p-eNOS ( p = 0.005) were found in siRNA group, compared with control group after mCRP treatment. At least three-time experiments were conducted. 3E. Different correlation analyses were conducted to examine the relationship between elevated peripheral mCRP and CD31 + microvessels in the brain. The graphs show that the length of CD31 + microvessels (y-axis) was positively associated with the CD31 level (r = 0.54, p < 0.0001) and the levels of PLA (ApoE-CD31) (r = 0.27, p = 0.03) but negatively associated with the levels of PLA (mCRP-CD31) (r = -0.65, p < 0.0001) and pCD31 (r = - 0.27, p = 0.04). Data are expressed as the mean ± SEM. Two-way ANOVA with Tukey’s post hoc test and Pearson or Spearman correlation test were applied. * p < 0.05, ** p < 0.01, *** p < 0.001. The scale bar is 50 µm.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Staining, Expressing, Comparison, Immunostaining, Biomarker Discovery, Knock-In, Western Blot, Transfection, Knockdown, Control

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: 4A and 4B. Representative images of i.p. mCRP-induced neuronal tau phosphorylation (pTau) in the cortex (A) and the CA3 region of the hippocampus (B) are shown. Double immunostaining of pTau (stained with PHF1, green) and neuronal marker (stained with NeuN, red); nuclei were stained with DAPI. Quantification of PHF1 levels in NeuN-positive cells showed statistical significance after i.p. mCRP in the cortex ( p = 0.006) and in the CA3 region ( p = 0.02) only in ApoE4, but not ApoE2 and ApoE3, mice. n =11-14 in each group. 4C. Representative images of CD68 + active microglia (red), Iba1 + microglia (green) and GFAP + astrocytes (purple) in the cortex of WT and ApoE knock-in mice after the i.p. treatment of PBS vs. mCRP are shown. Quantification of the microglial biomarkers CD68 ( p = 0.02) and Iba1 ( p = 0.0005) in the cortex showed significant differences between the PBS and mCRP groups only in ApoE4 mice, but the astrocyte biomarker GFAP ( p = 0.12) only showed tendency in the ApoE4 mice. n =11-14 in each group. 4D. Double immunostaining of CD8 (red), CD3 (green), and double-positive cells (yellow) was performed to study the transcytosis of T lymphocytes in the cortex after i.p. treatment with PBS (left columns) vs. mCRP (right columns) in WT and different ApoE knock-in mice. Quantification of CD8 + T lymphocytes and CD8 + /CD3 + T lymphocytes in the cortex and the comparisons between PBS vs. mCRP treatment in each genotype was conducted. mCRP significantly increased the number of T lymphocytes only in the WT ( p = 0.05) and ApoE4 ( p = 0.001) mice. n = 7-8 in each condition. 4E. Different correlation analyses using the data from all the mice used in the experiments were conducted to examine the relevance of different factors of the mCRP vs. ApoE to CD31 binding and the development of AD related pathology in the brain. The first row graphs show that the CD31 level (r = -0.48, p < 0.0001) was inversely related to PHF1 level in the brain, but PLA (ApoE-CD31) was not; the levels of PLA (mCRP-CD31) (r = 0.45, p = 0.02) and pCD31 (r = 0.40, p = 0.002) were positively correlated with PHF1 levels. The second row graphs show that the CD31 level (r = -0.35, p = 0.007) was inversely correlated with CD68/Iba1-positive cells in the brain, but PLA (ApoE-CD31) was not; the levels of PLA (mCRP-CD31) (r = 0.52, p = 0.02) and pCD31 (r = 0.45, p = 0.003) were positively correlated with double staining of CD68/Iba1 + cells. The third row graphs show that the number of CD8 + /CD3 + T lymphocytes (y-axis) was negatively associated with CD31 level (r = -0.28, p = 0.03) and positively associated with the levels of pCD31 (r = 0.54, p < 0.0001); however, there was no association with PLA (ApoE-CD31) or PLA (mCRP-CD31). The forth row graphs show that the length of CD31 + microvessels (x-axis) was negatively correlated with the levels of pTau (r = -0.56, p < 0.0001), the numbers of CD68 + /Iba1 + cells (r = -0.47, p = 0.0001), the numbers of CD8 + /CD3 + T lymphocytes (r = -0.48, p < 0.0001) and tended to be correlated with the levels of Aβ1-42 (r = -0.21, p = 0.06) in the brain. Data are expressed as the mean ± SEM. One or two-way ANOVA with Tukey’s post hoc test and Pearson or Spearman correlation test were applied. * p < 0.05, ** p < 0.01, *** p < 0.001. The scale bar: 50 µm.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Phospho-proteomics, Double Immunostaining, Staining, Marker, Knock-In, Biomarker Discovery, Binding Assay, Double Staining

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: The human temporal cortex of healthy controls (n = 8) and AD patients (n = 10) was used for immunostaining. ApoE4 carriers (red dots) and ApoE2 carriers (green dots) are illustrated. 5A. Representative images of CD31 + microvessels (red), mCRP (green) and the merged images (yellow) in the temporal cortex are shown. The levels of microvessel mCRP and mCRP-CD31 binding were quantified and compared. AD brains had significantly higher mCRP immunostaining in microvessels (yellow) than control brains ( p =0.01). AD brains tended to have lower levels of microvessel CD31 expression ( p = 0.09) and higher levels of mCRP-CD31 binding detected by using PLA ( p = 0.09) than control brains. 5B. PLA was also performed to detect interaction/binding between ApoE and CD31 in the temporal cortex of healthy controls and AD patients. Representative images of positive PLA (orange), ApoE (green) and CD31 (purple) staining are shown. The levels of microvessel ApoE and ApoE-CD31 were quantified and compared. AD brains had significantly lower levels of microvessel ApoE expression ( p = 0.02) and ApoE-CD31 binding ( p = 0.03) than control brains. 5C. Western blots for pCD31 and CD31 expression in the temporal cortex were performed and quantified after normalization against β-actin and compared between AD and controls. AD brains had higher levels of pCD31 ( p = 0.02), tended to have lower levels of total CD31 ( p = 0.07), and had a higher pCD31/CD31 ratio ( p = 0.03) than control brains. 5D. Double immunostaining of temporal cortex sections with CD31 (red), pCD31 (green) and the merged images (yellow) were conducted to examine CD31 phosphorylation and microvessel integrity. AD brains had higher brain pCD31 levels ( p = 0.05), pCD31/CD31 ratio ( p = 0.03), and microvessel pCD31 ( p = 0.01). Measurement of the lengths of CD31 + microvessels from 3D images revealed shorter lengths in AD brains than in controls ( p < 0.001). 5E. Correlation analyses were conducted to investigate the mCRP-ApoE-CD31 pathway and brain AD pathology in humans. In the first row, the microvessel pCD31 level (y-axis) was positively associated with mCRP (r = 0.67, p = 0.003) and mCRP-CD31 binding (PLA) (r = 0.53, p = 0.01) and negatively associated with ApoE-CD31 binding (PLA) (r = -0.49, p = 0.04). Microvessel length (y-axis) was negatively associated with pCD31 (r = - 0.55, p = 0.02) but positively associated with ApoE-CD31 (r = 0.69, p = 0.002). In the second row, the Braak stage (y-axis) was negatively associated with CD31 expression (r = -0.49, p = 0.04), ApoE-CD31 binding (r = -0.61, p = 0.007) and microvessl length (r = - 0.70, p = 0.001); in contrast, the Braak stage was positively associated with mCRP-CD31 binding (r = 0.51, p = 0.03) and microvessel pCD31 expression (r = 0.76, p = 0.0003). In the third row, the CAA (y-axis) was negatively associated with CD31 expression (r = - 0.50, p = 0.03), ApoE-CD31 binding (r = -0.66, p = 0.003) and microvessl length (r = - 0.67, p = 0.003); in contrast, CAA was not associated with the level of mCRP-CD31 binding but was positively associated with microvessel pCD31 expression (r = 0.54, p = 0.02). In the fourth row, the Mini-Mental State Exam (MMSE) score (y-axis) was positively associated with CD31 expression (r = 0.54, p = 0.03), the level of ApoE-CD31 binding (r = 0.52, p = 0.04) and microvessl length (r = 0.83, p < 0.0001); in contrast, MMSE score tended to be negatively associated with mCRP-CD31 binding (r = -0.47, p = 0.07) and significantly associated with the level of microvessel pCD31 + cells (r = -0.69, p = 0.003). Data are expressed as the mean ± SEM. Student’s t-test and Pearson or Spearman correlation tests were used. * p < 0.05, ** p < 0.01, *** p < 0.001. The scale bars are 100 µm, 20 µm, and 10 µm.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Immunostaining, Binding Assay, Control, Expressing, Staining, Western Blot, Double Immunostaining, Phospho-proteomics

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: 6A. The schematic showed CD31 + brain endothelial cells (BECs) were isolated by flow sorting and subjected to RNA sequencing from the mouse brain treated with i.p. PBS vs. mCRP. 6B-D. GSEA based on their differential gene expression was conducted to study the prominent pathways enriched in gene lists from different ApoE genotypes for AD pathogenesis. For the comparison between mCRP and PBS in each genotype, genes were ranked by GSEA based on their differential expression level. Four pathways, including 1) oxidative phosphorylation, 2) mitochondrial metabolism and respiration, 3) Alzheimer’s disease and 4) mTORC1 signaling, were upregulated by mCRP in ApoE4 mice but were downregulated in ApoE2 mice, while ApoE3 mice were in the middle (6B). In contrast, another four pathways, including 1) the histone lysine methyltransferase pathway, 2) synapse formation, 3) Notch signaling and 4) vasculogenesis, were upregulated by mCRP in ApoE2 mice but downregulated in ApoE4 mice, while ApoE3 mice were in the middle (6C.) The top or bottom of the ranked gene list in each ApoE comparison was evaluated using the enrichment score (green line) to determine whether a candidate pathway was significant. Black vertical lines mark positions where members of a particular pathway appear in the ranked list of genes. NES, normalized enrichment score; FDR, false discovery rate and q values. 6D shows the heatmaps of z-scored gene intensities of the differentially expressed genes enriched in the pathways/modules of oxidative phosphorylation and Alzheimer’s disease for ApoE2 and ApoE4 CD31 + BECs after the i.p. treatment. The key factors involved in the Alzheimer’s disease pathway that overlapped with the oxidative phosphorylation pathway were ranked and labeled by different intensities of yellow color. 6E. The RNAseq data were used for the functional enrichment analysis of the different signatures between ApoE4 and ApoE2 after the i.p. treatment of PBS vs. mCRP by using the ToppCluster tool (FDR correction, p < 0.05). The red/green box represents the top genes with up/downregulated expression in the ApoE4 mCRP vs PBS comparisons and down/upregulated expression in the ApoE2 mCRP vs PBS comparisons. The genes were connected to form a hub biological process and pathway (blue box). 6F. The CD31-related pathways are listed and shown according to NES comparison between mCRP vs PBS in each ApoE genotype (left panel). The network of CD31 and ApoE together with other components involved in biological processes, including vasculogenesis, leukocyte migration and Ras signaling pathways, is illustrated (right panel). Data are expressed as the mean ± SEM. One or two-way ANOVA with Tukey’s post hoc test was used. * p < 0.05, ** p < 0.01, *** p < 0.001; # p < 0.05, ## p < 0.01, ### p < 0.001.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Isolation, RNA Sequencing, Gene Expression, Comparison, Quantitative Proteomics, Phospho-proteomics, Labeling, Functional Assay, Expressing, Migration, Protein-Protein interactions

Journal: bioRxiv

Article Title: Competition between distinct ApoE alleles and mCRP for the endothelial receptor CD31 differentially regulates neurovascular inflammation and Alzheimer’s disease pathology

doi: 10.1101/2021.05.30.446344

Figure Lengend Snippet: This study demonstrated a novel pathological mechanism, the competition of ApoE and mCRP to CD31binding, for cerebrovascular neuroinflammation resulting in an early stage of AD pathogenesis in the brain. During the chronic stage of peripheral inflammation, pCRP proteins disassociate into mCRP. mCRP binds to CD31 on microvessels to increase CD31 phosphorylation (pCD31), cause damage to the cerebrovasculature and induce extravasation of T lymphocytes into the brain, leading to AD pathogenesis (ApoE4>ApoE3>ApoE2). This process is antagonized by ApoE-CD31 binding (ApoE2>ApoE3>ApoE4) to block mCRP-CD31 binding and differentially regulate pathways (mitochondrial function, epigenetics and vasculogenesis) to intervene in the neurodegenerative process of AD.

Article Snippet: To detect mCRP and CD31 interaction, samples were incubated overnight at 4°C with mouse anti-mCRP antibody (1:50, 3H12) and either goat anti-CD31 antibody (1:200, #AF3628, R&D systems, Minneapolis, MN, USA) for mouse samples or rabbit anti-CD31 antibody (1:200, #ab28364, Abcam, Cambridge, MA, USA) for human samples.

Techniques: Phospho-proteomics, Binding Assay, Blocking Assay