Journal: Stem Cell Research & Therapy

Article Title: Conditioned medium-preconditioned EPCs enhanced the ability in oligovascular repair in cerebral ischemia neonatal rats

doi: 10.1186/s13287-021-02157-4

Figure Lengend Snippet: Transplantation of EPCs promoted oligovascular remodeling in WMI rat. a Immunofluorescent staining showed EPC-derived CD31-positive ECs (green) integrated into the host brain and formed vascular-like structure, scale bar = 5 μm. The IOD of CD31 was quantified. b HI-induced OPC apoptosis was detected by PDGFRα immunofluorescent and TUNEL staining, scale bar = 20 μm. The percent of TUNEL and PDGFRα double-positive cells was analyzed. c MBP immunofluorescent staining and quantitative analysis were conducted for myelination evaluation, scale bar = 20 μm. d Representative electron microscopic images of the corpus callosum were shown. The g- ratio was calculated in each group. All values are expressed as the mean ± SD ( n = 4 for each group). * P < 0.05, ** P < 0.01, *** P < 0.001 vs. sham group; # P < 0.05, ## P < 0.01 vs. vehicle group; Δ P < 0.05 vs. EC-pEPCs group. Data were analyzed using Student’s t test

Article Snippet: For immunofluorescence, the brain sections were blocked with 10% goat serum and/or 0.3% Triton X-100 in 0.01 mol/l PBS for 40 min at 37 °C, followed by incubation with primary antibodies for MBP (1:1000, rabbit IgG, Abcam, Cambridge, UK) and platelet-derived growth factor receptor α (PDGFR-α, 1:1000, rabbit IgG, Abcam, Cambridge, UK), Claudin-5 (1:1000, rabbit IgG, Sigma), CD133 (rabbit IgG, 1:100, Abcam, USA), human CD31 (1:100, rabbit IgG, Sino Biology), and vWF (1:200, mouse IgG, Abcam, USA).

Techniques: Transplantation Assay, Staining, Derivative Assay, TUNEL Assay

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A . Pulmonary CT scans from SAVI patients (SAVI 1 and SAVI 5) showing fibrotic regions. Additional scans are shown in Figure S1A. B . Pathological characterization of SAVI lung tissue. Left panels: H&E and Masson-Trichrome staining of alveolar regions (SAVI 5) and controls (Ctrl), highlighting alveolar wall thickening and alveolar capillary fibrosis (orange box and magnified insets). Scale bar: 150 µm. Right panel: Quantification of inflammation and fibrosis scores (n=7 SAVI patients, n=5 controls). Mann-Whitney test; *, 0.01

Article Snippet: Subsequently, CD31 positive cells from the induction culture were enriched using CD31 magnetic beads (Miltenyi Biotec Cat#130091935), and then further seeded at approximately 2000 cells/cm2 on Corning® BioCoat® Collagen I plates (Corning, Cat#356450) and cultured in a 1:1 mixture of EGM2 growth media (EGM TM -2, Lonza, Cat#CC-3162) and Human Endothelial-SFM (Cat#11111044 Fisher Scientific).

Techniques: Staining, MANN-WHITNEY, Multiplex Assay, Control, Labeling, Fluorescence, Two Tailed Test, Double Staining, Gene Expression

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Experimental workflow and group design (created with BioRender.com ). B. Flow cytometric analysis of endothelial surface markers in HC, SAVI, and isogenic–SAVI (iso-SAVI) iECs. SAVI iECs progressively lost CD144 (VE-cadherin) and CD31 beginning at P3. Data summarize four HC- and SAVI-derived iEC lines and two iso-SAVI lines (mean ± SEM; ***p < 0.001, 2-way ANOVA). Representative flow cytometry profiles are shown in Figure S2C. C. Morphology and tube-formation capacity of iECs. Upper: HC and iso-SAVI iECs maintained cobblestone morphology from P1 to P5, whereas SAVI iECs transitioned to elongated fibroblast-like cells. Scale bar: 100 µm. Lower: Tube-formation assays performed at passages 1, 3, and 5; representative P5 images are shown (P1 images in Figure S2B). Total branch length and mesh area comparisons across groups are shown on the right (mean ± SEM; ***p < 0.001, **p < 0.01, *p < 0.05; 2-way ANOVA). Scale bar: 200 µm. D. Western blot analysis of endothelial (VE-cadherin, CD31) and mesenchymal (SMA, SM22) markers in iECs at P5. iECs were generated from three HC and three SAVI donors, with two iso-SAVI lines (three clones total). Representative blots and quantification (mean ± SEM; Mann–Whitney test) are shown. GAPDH served as a loading control. E. Schematic illustration summarizing panel D.

Article Snippet: Subsequently, CD31 positive cells from the induction culture were enriched using CD31 magnetic beads (Miltenyi Biotec Cat#130091935), and then further seeded at approximately 2000 cells/cm2 on Corning® BioCoat® Collagen I plates (Corning, Cat#356450) and cultured in a 1:1 mixture of EGM2 growth media (EGM TM -2, Lonza, Cat#CC-3162) and Human Endothelial-SFM (Cat#11111044 Fisher Scientific).

Techniques: Derivative Assay, Flow Cytometry, Western Blot, Generated, Clone Assay, MANN-WHITNEY, Control

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Top pathways enriched in SAVI P5 vs. P1 (iso-SAVI_SAVI cohort) by Ingenuity Pathway Analysis (IPA). Positive z-scores indicate pathway activation. Additional HC vs. SAVI analysis is shown in Figure S3A–B. B . EndMT signature heatmap in parental SAVI and isogenic iECs, with box-and-whisker plots of representative endothelial, mesenchymal, and previously reported EndMT-associated genes. Asterisks mark genes upregulated under TGFβ-induced EndMT but downregulated in SAVI iECs. C. IPA-derived transcription factor network of significant upstream regulators in the iso-SAVI_SAVI cohort (orange = activated; blue = inhibited). D . Heatmap of activation z-scores for the same transcription factors in panel C, showing similar patterns in SAVI vs. HC iECs and SAVI vs. HC lung tissue. E . Constitutive STAT3 nuclear translocation in SAVI iECs. STAT3 immunofluorescence (red) with DAPI (blue) at P5 from one SAVI and matched isogenic control line; white arrows indicate nuclear STAT3 (scale bar: 20 µm). Quantification reflects STAT3⁺ nuclei per total DAPI⁺ cells across 7–11 fields from 3–4 wells (mean ± SEM; *p < 0.05, two-tailed unpaired t-test). F . STAT3 activation in SAVI lung biopsies. Lung sections from 4 HC and 3 SAVI patients were stained for p-STAT3 Y705 (red) and CD31 (green) with DAPI (blue). Representative images (scale bar: 20 µm) and quantification of p-STAT3 Y705 /DAPI ratios (≥5 images per patient) show increased STAT3 activation in SAVI (mean ± SEM; ***p < 0.001, two-tailed unpaired t-test).

Article Snippet: Subsequently, CD31 positive cells from the induction culture were enriched using CD31 magnetic beads (Miltenyi Biotec Cat#130091935), and then further seeded at approximately 2000 cells/cm2 on Corning® BioCoat® Collagen I plates (Corning, Cat#356450) and cultured in a 1:1 mixture of EGM2 growth media (EGM TM -2, Lonza, Cat#CC-3162) and Human Endothelial-SFM (Cat#11111044 Fisher Scientific).

Techniques: Activation Assay, Whisker Assay, Derivative Assay, Translocation Assay, Immunofluorescence, Control, Two Tailed Test, Staining

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Workflow showing ATAC-seq, motif enrichment and pathway analyses. B . Heatmap of differentially accessible regions in HC and SAVI iECs at P1 and P3 (ATAC-seq; chi-square with Yates correction). C. Motif enrichment in regions differentially accessible between SAVI P1 and SAVI P3. Motifs enriched in the 43,995 SAVI P1–open regions appear on the left; motifs enriched in the 69 SAVI P3–open regions appear on the right. Additional comparisons are in Figure S5B. D. ATAC-seq in HLMECs stimulated with 2’3’-cGAMP or IFNβ for 8 h. Sankey plot shows the number of gained and lost accessible regions relative to non-treated (NT) cells. E. The enrichment of transcription factor binding motifs within the differentially accessible regions between the non-stimulated (NT), 2’3’-cGAMP or IFNβ stimulated HLMEC ATAC-seq libraries. Motif enrichment in regions more accessible after cGAMP stimulation in HLMECs (1,868 regions; none were more open in NT cells). Motif enrichment within the 1025 regions with increased accessibility in the IFNβ stimulated condition is shown on the right side of the y = 0 line. Cloud color as in B grey cloud indicates IRF TF motifs that are becoming increasingly accessible. F. These plots show the results of permutation tests from the regioneR package for the overlap between SOX18 ChIP-seq peaks from the Overman et al dataset and the SAVI P1 to SAVI P3 closing regions, or the HC P1 to HC P3 closing regions. A differential permutation test was also conducted to compare the relative enrichment of SOX18 ChIP-seq peaks in these two sets of regions. The green bar indicates the observed number of overlaps between the datasets. The black bar indicates the mean value of overlaps between the transcription factor ChIP-seq peaks and the random permuted regions. the distribution of overlaps from the permutations are shown as the gray histogram). The red bar indicates the number of overlaps at the threshold of significance p = 0.05. Differential enrichment analysis (bottom plot) showed significant overrepresentation of SOX18 binding sites in SAVI closing CRs at P3 (273 regions, z = 29.54 ; p < 0.001). G . The pathway enrichment of genes annotated by GREAT to differentially accessible regions from the SAVI iEC P1 to SAVI iEC P3 conditions that exhibited a decrease in accessibility in the P3 cells. These regions (and annotated genes) were filtered by the regions that overlapped SOX18, JUN, FOS, and GATA2 ChIP-seq peaks from the HUVEC ChIP-seq data sets listed in the methods section. H. Gene expression changes of endothelial lineage transcription factors across SAVI vs. iso-SAVI iECs (P1, P5) and cGAMP-treated vs. NT HLMECs (d1, d5) by RNAseq analysis. Red points indicate higher expression in SAVI or cGAMP conditions; endothelial TFs are starred. I. Reduced SOX18 protein in SAVI iECs at P5 compared with HC and iso-SAVI lines (3 individual iEC lines for each group), and partial restoration (∼50%) following STING inhibitor treatment (IFM35883, 2.5 μM, P2–P5) in SAVI iECs (SAVI1, n = 3). GAPDH served as loading control. Data are represented as mean ± SEM; ***p < 0.001, **p < 0.01, two-tailed unpaired t-test. J. SOX18 downregulation in HC iECs following 20 μg/ml 2’3’-cGAMP stimulation (30 min–24 h), peaking at 8 h (∼50% reduction). Quantification from three HC donors (mean ±SEM, ***p < 0.001, two-tailed unpaired t-test). K. SOX18 overexpression preserves endothelial surface markers CD144 and CD31 in SAVI iECs at P5. SAVI iECs were transduced at P2 with control or SOX18 cDNA; Flow cytometry analysis at P5 shows increased CD144⁺ and CD31⁺cells in SOX18 overexpression group. Data are represented as mean ± SEM; ***p < 0.001, **p < 0.01, two-tailed unpaired t-test. (n = 4, SAVI1 iEC line).

Article Snippet: Subsequently, CD31 positive cells from the induction culture were enriched using CD31 magnetic beads (Miltenyi Biotec Cat#130091935), and then further seeded at approximately 2000 cells/cm2 on Corning® BioCoat® Collagen I plates (Corning, Cat#356450) and cultured in a 1:1 mixture of EGM2 growth media (EGM TM -2, Lonza, Cat#CC-3162) and Human Endothelial-SFM (Cat#11111044 Fisher Scientific).

Techniques: Binding Assay, ChIP-sequencing, Gene Expression, RNA sequencing, Expressing, Control, Two Tailed Test, Over Expression, Flow Cytometry

Journal: bioRxiv

Article Title: STING–STAT3–SOX18 Axis Drives EndMT and Epigenetic Reprogramming in SAVI Lung Fibrosis

doi: 10.64898/2026.03.23.713256

Figure Lengend Snippet: A. Schematic of drug testing in the SAVI in-vitro disease model. B. Partial rescue of SAVI iEC EndMT by the FDA-approved JAK/STAT inhibitor Baricitinib. SAVI iECs were treated with Baricitinib (1 μM) from P2 to P3–4 (early harvest due to toxicity). Flow cytometry analysis of CD144 (VE-cadherin) and CD31 showed modest preservation of endothelial markers versus DMSO. Data from three SAVI donors (mean ± SEM; *p < 0.05; two-tailed t-test). Effects in cGAMP-treated HLMECs are shown in Fig. S6A–C. C. Impact of STING inhibition and FDA-approved antifibrotic drugs on EC surface markers. SAVI iECs were treated from P2 with IFM35883 (2.5 μM), pirfenidone (10 μM), nintedanib (1 μM), or DMSO. STING inhibition nearly fully preserved CD144 (VE-cadherin) and CD31, pirfenidone had no effect, and nintedanib markedly worsened EndMT. Representative flow cytometry profiles in Fig. S6E. Data from ≥3 experiments in the SAVI1 line (mean ± SEM; *****p < 0.0001; 2-way ANOVA). D. STING inhibition normalizes EndMT markers in SAVI iECs. SAVI iECs treated with IFM35883 (2.5 μM) or DMSO from P2–P5 were analyzed by Western blot. VE-cadherin and CD31 increased, while SMA and SM22 decreased with STING inhibition. Quantification from three experiments in SAVI1; representative blots shown (mean ± SEM; ***p < 0.001, **p < 0.01; two-tailed unpaired t-test).

Article Snippet: Subsequently, CD31 positive cells from the induction culture were enriched using CD31 magnetic beads (Miltenyi Biotec Cat#130091935), and then further seeded at approximately 2000 cells/cm2 on Corning® BioCoat® Collagen I plates (Corning, Cat#356450) and cultured in a 1:1 mixture of EGM2 growth media (EGM TM -2, Lonza, Cat#CC-3162) and Human Endothelial-SFM (Cat#11111044 Fisher Scientific).

Techniques: In Vitro, Flow Cytometry, Preserving, Two Tailed Test, Inhibition, Western Blot

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

Journal: Stem Cell Research & Therapy

Article Title: Mesenchymal stem/stromal cells overexpressing leukemia inhibitory factor (LIF) promote arteriogenesis and functional recovery in a mouse model of critical hindlimb ischemia

doi: 10.1186/s13287-025-04762-z

Figure Lengend Snippet: Pro-angiogenic potential of MSC_LIF in vivo. A Matrigel plugs containing MSC or MSC_LIF conditioned medium or 0.9% saline solution (- CTL) after excision of the mouse ventral area ( n = 4 animals per group). B Quantification of hemoglobin (Hb) content inside the plugs using Drabkin’s reagent. C Indirect immunofluorescence of sections of paraffin-embedded Matrigel plugs labeled with antibodies against the endothelial marker CD31 (red) and the smooth muscle cell marker αSMA (green). Cell nuclei stained with DAPI (blue). Images obtained by laser confocal microscopy. Scale bars = 100 μm. D Quantification of the mean diameter of capillaries (CD31 + vessels) by area. E Quantification of the mean diameter of arterioles (vessels simultaneously CD31/ αSMA + ) by area. The one-way ANOVA test and the Bonferroni post-test were used to analyze statistical differences. Values expressed as mean ± SEM of two independent experiments. *** p < 0.001; ** p < 0.01

Article Snippet: Tissues were stained with goat anti-mouse CD31 antibody (R&D Systems, diluted 1:100 in 1% BSA Triton-X 0.3% solution) overnight at 4 °C, followed by incubation with Alexa FluorTM 568-conjugated chicken anti-goat antibody (Invitrogen - Thermo Fisher Scientific, diluted 1:1000 in 1x PBS), and Alexa FluorTM 488-conjugated rat anti-mouse αSMA antibody (eBioscience - Thermo Fisher Scientific, diluted 1:400 in 1x PBS) for 1 h at room temperature.

Techniques: In Vivo, Saline, Immunofluorescence, Labeling, Marker, Staining, Confocal Microscopy

Journal: Stem Cell Research & Therapy

Article Title: Mesenchymal stem/stromal cells overexpressing leukemia inhibitory factor (LIF) promote arteriogenesis and functional recovery in a mouse model of critical hindlimb ischemia

doi: 10.1186/s13287-025-04762-z

Figure Lengend Snippet: Assessment of blood vessel distribution in the GST muscle. A Indirect immunofluorescence of cryopreserved sections of GST muscle stained with antibodies against the endothelial marker CD31 (yellow) and the smooth muscle cell marker SMA (green). Cell nuclei were stained with DAPI (blue), and muscle cells were stained with phalloidin (red) ( n = 5 animals per group). Images obtained by laser confocal microscopy. Scale bars = 100 μm. B Quantification of mean capillary length/diameter (CD31 + vessels) per area. C Quantification of mean arteriole length/diameter (vessels simultaneously CD31/αSMA+). The Kruskal-Wallis test and the Dunn’s Multiple Comparison post-test were used to analyze statistical differences. Values expressed as mean ± SEM of three independent experiments. *** p < 0.001; ** p < 0.01; * p < 0.05. The NAIVE group corresponds to the non-induced paw

Article Snippet: Tissues were stained with goat anti-mouse CD31 antibody (R&D Systems, diluted 1:100 in 1% BSA Triton-X 0.3% solution) overnight at 4 °C, followed by incubation with Alexa FluorTM 568-conjugated chicken anti-goat antibody (Invitrogen - Thermo Fisher Scientific, diluted 1:1000 in 1x PBS), and Alexa FluorTM 488-conjugated rat anti-mouse αSMA antibody (eBioscience - Thermo Fisher Scientific, diluted 1:400 in 1x PBS) for 1 h at room temperature.

Techniques: Immunofluorescence, Staining, Marker, Confocal Microscopy, Comparison

Journal: Journal of radiation research

Article Title: Hypoxia expression in radiation-induced late rectal injury.

doi: 10.1269/jrr.07099

Figure Lengend Snippet: Fig. 4. Double immunofluorescence staining in fibrotic region of irradiated rectal tissue. (A), HIF-1α and VEGF; (B), HIF-1α and CD31. HIF-1α was labeled with red color; VEGF and CD31 were labeled with green color and Merge was shown by yellow color. Cont: unirradiated control mice. Magnification: × 100.

Article Snippet: Briefly, the slides were treated with rabbit anti-TGF-β1 polyclonal antibody (1:150, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-HIF-1α polyclonal antibody (1:150, Santa Cruz Biotechnology), rabbit anti-VEGF polyclonal antibody (1:150, Santa Cruz Biotechnology) or rabbit anti-PECAM-1 (CD31) polyclonal antibody (1:150, Santa Cruz Biotechnology) as a primary antibody for overnight at 4°C.

Techniques: Double Immunofluorescence Staining, Irradiation, Labeling, Control

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Melanocortin 1 Receptor Regulates Pathological and Physiological Cardiac Remodeling

doi: 10.1161/JAHA.124.037961

Figure Lengend Snippet: A , Immunostaining of MC1R in cardiac cross‐section of C57Bl/6J mouse. In THE control section, anti‐MC1R antibody was replaced by purified normal rabbit IgG (isotype control). Scale bar 20 μm. B , Immunofluorescence staining of MC1R (red) and sarcomeric α‐actinin, α‐SMA or CD31 (green) in cardiac cross‐section of C57Bl/6J mouse. Scale bar 20 μm. C and D , Representative western blots and quantification of MC1R protein expression in the left ventricle of C57Bl/6J mice subjected to TAC for 4 or 8 wks. n=6–7 mice per group. *** P <0.001 and **** P <0.0001 vs sham by unpaired Student's t test. E and F , Quantitative real‐time polymerase chain reaction analysis of MC1R mRNA expression (normalized to the geometric mean of GAPDH and RPS18 ) in human induced pluripotent stem cell‐derived cardiomyocytes that were mechanically stretched for 24 or 48 h or treated with ET‐1 (100 nM) for 24 h. n=3–4 individual experiments/batches of differentiation. * P <0.05 and ** P <0.01 vs control by 2‐way ANOVA and Šídák's post hoc test ( E ) or Mann–Whitney U test ( F ). Data are mean±SEM. α‐SMA indicates α‐smooth muscle actin; ET‐1, endothelin 1; MC1R, melanocortin 1 receptor; and TAC, transverse aortic constriction.

Article Snippet: For immunofluorescence, heart sections were stained with primary antibodies against MC1R and sarcomeric α‐actinin (Merck Life Science; No. A7811), α‐smooth muscle actin (α‐SMA, Merck Life Science; No. A5228) or CD31 (R&D Systems; No. AF3628) followed by detection with fluorochrome‐conjugated secondary antibodies (Alexa Fluor 488 and Alexa Fluor 647; Jackson ImmunoResearch, West Grove, PA).

Techniques: Immunostaining, Control, Purification, Immunofluorescence, Staining, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, MANN-WHITNEY

Journal: Advanced Science

Article Title: Immune and Angiogenic Profiling of Mesenchymal Stem Cell Functions in a Subcutaneous Microenvironment for Allogeneic Islet Transplantation

doi: 10.1002/advs.202411574

Figure Lengend Snippet: MSC induction of functional vasculature development in NICHE. Quantification of VEGF in the cell reservoir of control ( n = 3–4/timepoint) and MSC‐loaded ( n = 5/timepoint) NICHE devices implanted for 2, 4, and 6 weeks in A) males and B) females. Protein levels were normalized to total protein content of the tissue homogenates. Mean ± SD, two‐way ANOVA with Bonferroni's multiple comparisons test (* p < 0.05; ** p < 0.01; *** p < 0.001). Representative immunofluorescent staining of NICHE vasculature in C) males and D) females at 4 weeks post‐implantation stained with functional blood vessel markers CD31 (red), eNOS (gold) and VE‐Cadherin (magenta). RBCs are autofluorescent in FITC channel (green). Scale bars, 50 µm. Fluorescence intensity analysis of E,H) CD31, F,I) eNOS, and G,J) VE‐Cad as relative expression in NICHE‐MSC compared to control devices at each timepoint for males and females ( n = 4 biological replicates; n = 4 fields of view per sample). Scatter plots show mean of all captured FOV ( n = 16), un‐paired Student's t‐test of averaged FOV per sample at each timepoint (* p < 0.05, ** p < 0.01, *** p < 0.001), denoting level of significance compared to control hydrogel only‐NICHE.

Article Snippet: [ ] (2) Blood vessel density = Vessel number FOV area (3) Vessel area % = Area occupied by vessels Total section area x 100 For immunofluorescence staining, following deparaffinization, rehydration, pressure‐cooker‐based antigen retrieval, and blocking with 5% goat serum in 0.1% BSA/TBS, sections were incubated with primary antibodies CD31 (NB100‐2284, Novus Biologicals, 1:200), VE Cadherin (36‐1900, Invitrogen, 1:25), and eNOS (ab300071, Abcam, 1:50) diluted in 1% BSA, 1% horse serum, 0.3% TritonX‐100, and 0.01% sodium azide in 1× PBS.

Techniques: Functional Assay, Control, Staining, Fluorescence, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Sesquiterpene Lactone Deoxyelephantopin Isolated from Elephantopus scaber and Its Derivative DETD-35 Suppress BRAF V600E Mutant Melanoma Lung Metastasis in Mice

doi: 10.3390/ijms22063226

Figure Lengend Snippet: Effect of DET and DETD-35 on various protein markers in the lungs of metastatic melanoma mice. A – E : The representative images of ( A ) Mel-A, ( B ) Ki67, ( C ) cleaved caspase-3, ( D ) N -cadherin, and ( E ) VEGF expression in lung tissues from sham, tumor control, DET- (20 mg/kg) and DETD-35- (20 mg/kg) treated mice. The quantification data were analyzed by IHC profiler plugin, ImageJ. The percentage of positive-staining intensity for each specific protein categorized as high positive, positive, and low positive/negative are summarized. Data are mean ± SD, n = 4. F – G : The representative immunofluorescence images of ( F ) CD31 (green) and COX-2 (red), ( G ) Neutrophils (NE, red), macrophages (F4/80, green) and M2-like macrophages (CD163, green) in lung tissues from sham, tumor control, DET- (20 mg/kg) and DETD-35- (20 mg/kg) treated mice. The quantification was performed using ImageJ software. Data are mean ± SD, n = 4. Means with significant differences are denoted with different letters (one-way ANOVA, p ≤ 0.05). ( H ) The representative image of oxidative stress marker 8-OHdG in the tumor nodules of lungs from tumor control, DET- (20 mg/kg) and DETD-35- (20 mg/kg) treated mice. The quantification data were analyzed by IHC profiler plugin, ImageJ. The percentage of positive-staining intensity for 8-OHdG categorized as high positive, positive, and low positive/negative is summarized. Data are mean ± SD, n = 4. The scale bar represents 50 µm.

Article Snippet: For immunofluorescence staining of lung tissues from the A375LM5 IF4g/Luc metastatic melanoma mouse model, the primary antibody against Neutrophil elastase (ab68672, Abcam, 1:200), CD31 (11265-1-AP, Proteintech, 1:200), CD163 (16646-1-AP, 1:200), F4/80 (cat. #123101, Biolegend, 1:200), COX-2 (sc-19999, Santa Cruz, 1:200) were probed with Alexa Fluor 488 or Alexa Fluor 594 conjugated secondary antibody (1:1000 dilution) (Bio-Rad Laboratories, Hercules, California, USA).

Techniques: Expressing, Control, Staining, Immunofluorescence, Software, Marker

Journal: Stem Cell Research & Therapy

Article Title: Progenitor-like cells derived from mouse kidney protect against renal fibrosis in a remnant kidney model via decreased endothelial mesenchymal transition

doi: 10.1186/s13287-015-0241-8

Figure Lengend Snippet: MKPC injection preserves capillary density and prevents EndoMT in five-sixths nephrectomized mice. a – c Capillary rarefaction occurs in five-sixths nephrectomized mice treated with saline a , but MKPC injection preserves capillary density b at 17 weeks after establishment of chronic kidney injury. Scale bar: 50 μm. d – i Confocal microscopy demonstrates α-SMA ( red ) and CD31 ( green ) staining in 17-week nephrectomized mice. Arrows indicate α-SMA and CD31 double-positive cells in the capillaries. EndoMT occurs in the interstitium of cortex and cortico-medullary junction from five-sixths nephrectomized mice treated with saline. The MKPC injection prevents EndoMT. Scale bar: 50 μm h , 20 μm i . α-SMA alpha-smooth muscle actin, Junction cortico-medullary junction of the kidney, MKPC mouse kidney progenitor-like cells, Saline five-sixths nephrectomized mice treated with saline

Article Snippet: In immunofluorescence, mouse anti-human smooth muscle actin (Dako Cytomation, Carpinteria, CA , USA) and rabbit anti-mouse CD31 (Santa Cruz, Texas, USA) were used as primary antibodies.

Techniques: Injection, Saline, Confocal Microscopy, Staining

Journal: Stem Cell Research & Therapy

Article Title: Progenitor-like cells derived from mouse kidney protect against renal fibrosis in a remnant kidney model via decreased endothelial mesenchymal transition

doi: 10.1186/s13287-015-0241-8

Figure Lengend Snippet: MKPC conditioned medium promote angiogenic process in vitro. a , b Conditioned medium from MKPCs ameliorates endothelial cell death following hypoxic culture condition. Endothelial cells (MMECs) were treated with or without conditioned medium from MKPCs ( MKPC CM ) under normoxic or hypoxic culture conditions for 24 or 28 hours. Cell death was measured by Annexin V and PI staining detected by flow cytometry. Each experiment was repeated three times. Cells in the upper left and upper right plots were considered death cells. c – f Conditioned medium from MKPCs prevents TGF-β-induced EndoMT in vitro. Confocal microscopy demonstrates DAPI ( blue ), CD31 ( green ), and α-SMA ( red ) in MMECs cultured with or without MKPC CM and with or without TGF-β for 14 days c . Representative western blot and relative bar graph analysis of CD31, α-SMA d , e , Smad3 and pSmad3 f protein level in MMECs after various treatments. Fibroblast-like change of MMECs was observed following TGF-β treatment, but the change was reduced in MMECs incubated with MKPC CM c . TGF-β treatment (5 ng/ml) caused increased α-SMA protein expression and decreased CD31 protein expression d , e . MKPC CM inhibited phosphorylation of Smad3 in MMEC after 1 hour of TGF-β stimulation f and attenuated these TGF-β-induced EndoMT responses. * P <0.05 compared with control or between two groups. α-SMA alpha-smooth muscle actin, DAPI 4′,6-diamidino-2-phenylindole, MMEC mouse pancreatic microvascular endothelial cell, TGF-β transforming growth factor beta

Article Snippet: In immunofluorescence, mouse anti-human smooth muscle actin (Dako Cytomation, Carpinteria, CA , USA) and rabbit anti-mouse CD31 (Santa Cruz, Texas, USA) were used as primary antibodies.

Techniques: In Vitro, Staining, Flow Cytometry, Confocal Microscopy, Cell Culture, Western Blot, Incubation, Expressing, Phospho-proteomics, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Single-Cell RNA Sequencing of Coronary Perivascular Adipose Tissue From End-Stage Heart Failure Patients Identifies SPP1 + Macrophage Subpopulation as a Target for Alleviating Fibrosis

doi: 10.1161/ATVBAHA.123.319828

Figure Lengend Snippet: Atherosclerosis was accompanied with the activation of endothelial cell (EC) and smooth muscle cell (SMC) subclusters in coronary perivascular adipose tissue (PVAT). A , A Uniform Manifold Approximation and Projection (UMAP) plot of all ECs, mesothelial cells, and SMCs (EMSs) colored according to cluster. B , Relative expression of classical markers in EMS cells. C , Mean expression of top marker genes in EMS clusters. D , The top 5 enriched gene ontology biological process terms of each EMS subcluster. E , The ratio of each EMS subcluster in the different phases. F , The expression level and regulon activity of TFs (transcription factors) in EMS subpopulations. G , Immunofluorescence staining of SOCS3 (red) and ACTA2 (green) in coronary PVAT in different phases. Scale bar, 100 µm. H , Quantification of ( G ) cell ratio per image (n=16 in nonatherosclerosis control [NC], n=14 in nonobstructive coronary atherosclerosis [NOCA], and n=10 in obstructive coronary atherosclerosis [OCA]). I , Immunofluorescence staining of DARC (red) and CD31 (green) in coronary PVAT in different phases. Scale bar, 100 µm. J , Quantification of ( I ) cell ratio per image (n=16 in NC, n=14 in NOCA, and n=10 in OCA). Mann-Whitney U test was performed to compare the cellular ratio between each 2 groups; P values were adjusted for multiple hypothesis testing using the Benjamini-Hochberg method. ACTA2 indicates actin alpha 2; DARC, duffy antigen receptor for chemokines; MC, mesothelial cell; and SOCS3, suppressor of cytokine signaling 3.

Article Snippet: The primary antibodies used were as follows: anti-SPP1 (secreted phosphoprotein 1; ab218237, 4 μg/mL; Abcam), anti-ACTA2 (actin alpha 2; ab7817, 10 μg/mL; Abcam), anti-CD68 (ZM-0060, 10 μg/mL; ZSGB-BIO), anti-SOCS3 (suppressor of cytokine signaling 3; ab280884, 6 μg/mL; Abcam), anti-COL1 (collagen I; ab138492, 8 μg/mL; Abcam), anti-DARC (duffy antigen receptor for chemokines; ab137044, 6 μg/mL; Abcam), anti-CD31 (ZA-0568, 6 μg/mL; ZSGB-BIO), anti-APOE (18254-1-AP, 6 μg/mL; Proteintech), and anti-DCN (decorin; ab268048, 2 μg/mL; Abcam).

Techniques: Activation Assay, Expressing, Marker, Activity Assay, Immunofluorescence, Staining, MANN-WHITNEY

Journal: Life

Article Title: Inhibition of Pathological Increased Matrix Metalloproteinase (MMP) Activity for Improvement of Bone Regeneration in Diabetes

doi: 10.3390/life12020134

Figure Lengend Snippet: Immunohistochemical staining of CD31 (platelet endothelial cell adhesion molecule 1, PECAM-1. Diminished angiogenesis in regenerating bone of mice with diabetes was slightly reconstituted by inhibition of MMP activity. Values are depicted as ±SD. p -value: ** < 0.01. Scale bar: 50 μm.

Article Snippet: For immunofluorescent stainings, primary antibodies against CD31 (rat, monoclonal, BD Biosciences, 553370, 1:400), MMP3 (rabbit, monoclonal, abcam, ab52915, 1:100), and collagen type I alpha 1 (Col1A1, mouse, monoclonal, SantaCruz Biotechnology, sc-293182, 1:100) were utilized.

Techniques: Immunohistochemical staining, Staining, Inhibition, Activity Assay