Journal: Nature Materials

Article Title: Parenchymal and stromal tissue regeneration of tooth organ by pivotal signals reinstated in decellularized matrix

doi: 10.1038/s41563-019-0368-6

Figure Lengend Snippet: Fig. 4 | Alx3-restored adult MSC-induced angiogenesis and VEGF signalling. a,d, Angiogenesis in regenerated tissues with magnifications. d, native dentin. b,e, Human von Willebrand factor immunohistochemistry with magnifications. Green arrowheads, endothelial cells of human origin (e); black arrowheads, endothelial cells of host (mouse) origin (e); red arrowheads chimeric blood vessels (e). c,f, Human mitochondria staining; red arrowheads, human cells; black arrowheads, host (mouse) cells. a–f, n = 5 independent biological samples. g, Blood vessel (bv) quantification. n = 5 independent biological samples, presented as the mean ± s.d. P values calculated by one-way ANOVA with Bonferroni: **P < 0.01, ***P < 0.001. h, CCK8 of HUVECs treated with conditioned medium (CM) by vector control or Alx3-restored MSCs. n = 4 independent biological samples, presented as the mean ± s.d. P values calculated by one-way ANOVA with Bonferroni: *P < 0.05). i–o, Transwell migration assay by Alx3 conditioned medium or with neutralizing antibody or VEGFR2 inhibitor (i–n) and quantification (o). n = 4 independent biological samples, presented as mean ± s.d. P values calculated by one-way ANOVA with Bonferroni: *P < 0.05, **P < 0.01, ***P < 0.001). p, Mouse VEGF A promoter luciferase reporter with two Alx3-binding elements in the dashed red boxes (left) and VEGF luciferase assay on Alx3 transfection (right). n = 3 independent biological samples, presented as median with range. P values calculated by Kruskal–Wallis tests: *P < 0.05). BE, binding element. q,r, VEGF production and p-VEGFR2 activation by western blot (q) and quantification (r). n = 4 independent biological samples, presented as the mean ± s.d. P values calculated by one-way ANOVA with Bonferroni: *P < 0.05, **P < 0.01, ***P < 0.001. s–x: The tube formation of HUVECs treated by Alx3 conditioned medium with or without NAb or VEGFR2 inhibitor. n = 3 independent biological samples. y, Tube length quantification. n = 3 independent biological samples, presented as the median with range. P values calculated by Kruskal–Wallis tests: *P < 0.05, **P < 0.01. z, rtPCR of VE-cadherin, PECAM1, VEGF and Flk1. n = 4 independent biological samples, presented as the mean ± s.d. P values calculated by one-way ANOVA with Bonferroni: *P < 0.05, **P < 0.01, ***P < 0.001).

Article Snippet: The 1.6 kB VEGF luc plasmid was a gift from P. D’Amore (Addgene plasmid no. 29667).

Techniques: Immunohistochemistry, Staining, Plasmid Preparation, Control, Transwell Migration Assay, Luciferase, Binding Assay, Transfection, Activation Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Cells

Article Title: Vitamin D Receptor Expression Limits the Angiogenic and Inflammatory Properties of Retinal Endothelial Cells

doi: 10.3390/cells12020335

Figure Lengend Snippet: Isolation and characterization of mouse retinal EC. Vdr +/+ and Vdr −/− retinal EC were prepared and cultured as described in Materials and Methods. ( A ) Morphology of cells at subconfluent and confluent. ( B ) Vitamin D receptor expression levels were accessed by qPCR analysis (n = 3, *** p < 0.001). ( C ) Cells were incubated with medium alone (cells), 0.41% ethanol (vehicle), or 10 µM calcitriol for 24 h and VDR levels were measured by Western blot analysis. ( D ) Expression levels of EC markers were determined by flow cytometry. VE-cad; VE-cadherin and PECAM-1; Platelet endothelial cell adhesion molecule-1. Relative median fluorescent intensities are indicated in the top left corner of each panel. These cells were also positive for B4-lectin (a mouse EC specific lectin) and lacked the expression of PDGFRβ and smooth muscle actin (pericyte markers; not shown).

Article Snippet: The primary antibodies used were rat anti-PECAM-1 (553370; BD Bioscience) and rat anti-VE-cadherin (550548; BD Bioscience).

Techniques: Isolation, Cell Culture, Expressing, Incubation, Western Blot, Flow Cytometry

Journal: Cells

Article Title: Vitamin D Receptor Expression Limits the Angiogenic and Inflammatory Properties of Retinal Endothelial Cells

doi: 10.3390/cells12020335

Figure Lengend Snippet: Cellular localization and expression levels of ZO-1, N-cadherin, VE-cadherin, p120-catenin, and β-catenin. ( A ) Retinal EC were cultured on fibronectin-coated chamber slides to confluence and stained with specific antibodies as described in . ( B , C ) Western blot analysis of junctional proteins (n = 3–4, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001).

Article Snippet: The primary antibodies used were rat anti-PECAM-1 (553370; BD Bioscience) and rat anti-VE-cadherin (550548; BD Bioscience).

Techniques: Expressing, Cell Culture, Staining, Western Blot

Journal: Scientific Reports

Article Title: Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail

doi: 10.1038/s41598-017-03532-z

Figure Lengend Snippet: Low shear stress induced mesenchymal genes via Snail. ( a ) HUVEC were exposed to low oscillatory (+/−4 dyn/cm 2 ) or high (13 dyn/cm 2 ) wall shear stress (WSS) using a parallel plate system. ( b ) PAEC were exposed to orbital flow to generate low (5 dyn/cm 2 ) or high (15 dyn/cm 2 ) wall shear stress. ( a , b ) After 72 h, levels of EndMT marker transcripts and VE-cadherin transcripts were quantified by qRT-PCR. The expression level at the low WSS site is presented relative to the expression at the high WSS site (normalised to 1; dotted line). Data were pooled from six independent experiments using cells from different donors and mean levels +/− SEM are shown. ( c–e ) HUVEC were exposed to orbital flow to generate low (5 dyn/cm 2 ) or high (15 dyn/cm 2 ) WSS for 72 h. ( c ) Expression of N-cadherin (green) and VE-cadherin (red) was determined by immunofluorescent staining and co-staining using DAPI (blue). Scale bar, 50 μm. The proportion of cells that expressed N-cadherin or VE-cadherin was measured. ( d ) The expression levels of N-cadherin (left) and VE-cadherin (right) were assessed by Western blotting using specific antibodies and anti-PDHX antibodies were used to control for total protein levels. Representative blots are shown. Bands were quantified by densitometry. ( e ) Expression of Snail (green) was determined by immunofluorescent staining and co-staining using DAPI (blue). Scale bar, 50 μm. Fluorescence intensity was quantified in multiple cells. ( f ) HUVEC were transfected with siRNA targeting Snail or with scrambled sequences and exposed to orbital flow for 72 h. Cells exposed to low WSS (5 dyn/cm 2 ) were collected and transcript levels of Slug, N cadherin and α-SMA were quantified by qRT-PCR. ( c – f ) Data were pooled from three independent experiments using cells from different donors and mean levels +/− SEM are shown.

Article Snippet: Antibodies targeting human and murine Snail (ab180714, Abcam), human N-cadherin (ab12221, Abcam), human VE-cadherin (555661, BDPharmingen), murine CD31 (102514, Biolegend) and Ki67 (ab15580, Abcam) were obtained commercially.

Techniques: Marker, Quantitative RT-PCR, Expressing, Staining, Western Blot, Fluorescence, Transfection

Journal: Scientific Reports

Article Title: Shear stress induces endothelial-to-mesenchymal transition via the transcription factor Snail

doi: 10.1038/s41598-017-03532-z

Figure Lengend Snippet: Snail was preferentially expressed at low shear atherosusceptible sites. ( a ) EC were freshly-isolated from low wall shear stress (WSS; inner curvature) and high WSS (outer curvature) regions of the aorta in six pigs. Levels of Snail, Slug, N-cadherin, α-SMA and VE-cadherin mRNA were quantified by qRT-PCR. The expression level at the low WSS site is presented relative to the expression at the high WSS site (normalised to 1; dotted line). Mean levels +/− SEM are shown. ( b , c ) EC at low WSS (susceptible) or high WSS (protected) regions of the aorta were studied by en face staining. ( b ) C57BL/6 mice (n = 5) were stained using anti-Snail antibodies (red), co-stained using anti-CD31 antibodies (green) and counterstained using TO-PRO-3 (DNA; blue). ( c ) TWIST1 cKO or TWIST1 fl/fl mice (n = 4 each group) were stained using anti-Snail antibodies (green) and counterstained using TO-PRO-3 (DNA; blue). Representative images (scale bar, 10 μm) and quantitation of Snail fluorescence levels (mean +/− SEM) are shown. Differences between means were assessed using a paired t-test ( b ) or two-way ANOVA ( c ).

Article Snippet: Antibodies targeting human and murine Snail (ab180714, Abcam), human N-cadherin (ab12221, Abcam), human VE-cadherin (555661, BDPharmingen), murine CD31 (102514, Biolegend) and Ki67 (ab15580, Abcam) were obtained commercially.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Staining, Quantitation Assay, Fluorescence