Journal: bioRxiv

Article Title: Structure of EPCR in a non-canonical conformation

doi: 10.1101/2021.12.16.472967

Figure Lengend Snippet: (A) The canonical (left) and non-canonical EPCR structures are shown in grey and paleyellow colors, respectively. The residues in the α2 helix with severe folding transitions are highlighted. 2Fo-Fc electron density maps are shown as blue meshes. In order to confirm the novel conformation, maps were generated using either the canonical or non-canonical coordinates of residues Tyr154 through Thr157. The bound phospholipids are shown in the central cavities in stick format (B) Superposition of the canonical and non-canonical structures. Tyr154, Asn155 and Thr157 are highlighted for comparison purposes. (C) Directional switch in the α2 helix of EPCR.

Article Snippet: Human EPCR cDNA (Genscript) was PCR amplified and cloned in frame with a GP64 signal peptide in a pAcGP67A transfer vector, using BamHI and NotI restriction enzymes and OptizymeTM T4 DNA ligase (Thermo Fisher Scientific).

Techniques: Generated, Comparison

Journal: bioRxiv

Article Title: Structure of EPCR in a non-canonical conformation

doi: 10.1101/2021.12.16.472967

Figure Lengend Snippet: (A) Structure of the protein C Gla domain in complex with the canonical conformation of EPCR (PDB 1LQV). The contacts with the Gla domain established by EPCR Tyr154 are highlighted with grey dashed lines. Tyr154 residues in both the canonical and non-canonical EPCR structures are highlighted in sticks for comparison purposes and to better visualize the impact of the folding transition in protein C binding. (B) Upper panel, measurement of kinetic constant rates and affinity interaction between wild type EPCR and APC (upper panel). Red color traces denote buffer signal-substracted raw binding data and black traces indicate fitting to a 1:1 binding kinetic model. Lower panel, comparison of binding signal of 125 nM APC to EPCR or EPCR Y154A . (C) Upper panel, intermolecular contacts between the non-canonical EPCR Tyr154 and N-acetylglucosamine (NAG) in a crystallographic symmetry mate EPCR molecule. Lower panel, analogous view with the canonical EPCR structure (PDB 1L8J). The 1L8J symmetry mate (palecyan color) is shown superposed with the symmetry mate of the non-canonical EPCR structure.

Article Snippet: Human EPCR cDNA (Genscript) was PCR amplified and cloned in frame with a GP64 signal peptide in a pAcGP67A transfer vector, using BamHI and NotI restriction enzymes and OptizymeTM T4 DNA ligase (Thermo Fisher Scientific).

Techniques: Comparison, Binding Assay

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