Journal: Bioactive Materials

Article Title: Electrochemically derived nanographene oxide activates endothelial tip cells and promotes angiogenesis by binding endogenous lysophosphatidic acid

doi: 10.1016/j.bioactmat.2021.07.007

Figure Lengend Snippet: NGO activates endothelial tip cells via the LPAR6-Hippo-YAP signalling pathway. (a) Gene ontology analysis revealed the enrichment of biological processes, such as cell differentiation, angiogenesis and GPCRs, and cellular components involving the cytoskeleton. (b) The Hippo signalling pathway was enriched in the differentially expressed genes according to KEGG analysis. (c) The mRNA expression levels of LPAR1-6 in HUVECs treated with 0 or 5 μg/mL NGO for 24 h in RNA-seq detection. (d) Laser confocal microscopy showing that LPAR6 expression was upregulated in areas containing BSA-FITC labelled NGO. Scale bar, 20 μm. (e) Protein levels of LPAR6, RhoA, ROCK1, Lats1, p-YAP Ser127 and YAP1 in HUVECs treated with 0 or 5 μg/mL for 24 h were measured by western blotting. (f) Treatment with siRNA targeting YAP (200 nM) caused downregulation of the protein levels of KDR, DLL4, and CD34. (g) Wound healing assay of HUVECs treated with or without siYAP for 24 h in the NGO group. The wound area (%) was measured using ImageJ. (h) A tube formation assay was performed with HUVECs treated with siControl or siYAP and stimulated with NGO for 24 h. Quantitative analysis of the master segment length (μm) and numbers of nodes and meshes in the endothelial network. Data represent the mean ± SD (n = 3). * p < 0.05.

Article Snippet: After acclimation to the cages for 1 week, thirty-nine rats were randomly divided into the following four groups (n = 6): control (surgery without GelMA implantation), GelMA (GelMA implantation), NGO/GelMA (implantation of 0.1/0.5/1/2/5/10 wt% NGO/GelMA) and NGO/GelMA + Yes-associated protein (YAP) inhibitor (0.5 wt% NGO/GelMA + 10 μM verteporfin, Selleckchem).

Techniques: Cell Differentiation, Expressing, RNA Sequencing Assay, Confocal Microscopy, Western Blot, Wound Healing Assay, Tube Formation Assay

Journal: Bioactive Materials

Article Title: Electrochemically derived nanographene oxide activates endothelial tip cells and promotes angiogenesis by binding endogenous lysophosphatidic acid

doi: 10.1016/j.bioactmat.2021.07.007

Figure Lengend Snippet: The role of YAP in NGO-promoted angiogenesis in vivo. (a) HE staining and (b) CD31 immunohistochemical staining of the defective region 2 weeks after implantation in the NGO/GelMA groups with or without the YAP inhibitor verteporfin (10 μM). Scale bar, 100 μm. (c) 3D images of angiogenesis in the area of the calvarial defect 2 weeks after surgery. (d) CD31 and Emcn IF double staining revealing newly formed H-type blood vessels in the bone defect region (Scale bar, 50 μm), and the percentages of H-type blood vessels 2 weeks after surgery in rats treated with or without verteporfin were quantified. Data represent the mean ± SD (n = 6). *** p < 0.0001.

Article Snippet: After acclimation to the cages for 1 week, thirty-nine rats were randomly divided into the following four groups (n = 6): control (surgery without GelMA implantation), GelMA (GelMA implantation), NGO/GelMA (implantation of 0.1/0.5/1/2/5/10 wt% NGO/GelMA) and NGO/GelMA + Yes-associated protein (YAP) inhibitor (0.5 wt% NGO/GelMA + 10 μM verteporfin, Selleckchem).

Techniques: In Vivo, Staining, Immunohistochemical staining, Double Staining

Journal: Bioactive Materials

Article Title: Electrochemically derived nanographene oxide activates endothelial tip cells and promotes angiogenesis by binding endogenous lysophosphatidic acid

doi: 10.1016/j.bioactmat.2021.07.007

Figure Lengend Snippet: Schematic representation of the mechanisms by which NGO promotes early angiogenesis at the bone defect site. NGO-bound endogenous LPA induces the nuclear translocation of YAP, thereby activating tip cell specialization and promoting angiogenesis.

Article Snippet: After acclimation to the cages for 1 week, thirty-nine rats were randomly divided into the following four groups (n = 6): control (surgery without GelMA implantation), GelMA (GelMA implantation), NGO/GelMA (implantation of 0.1/0.5/1/2/5/10 wt% NGO/GelMA) and NGO/GelMA + Yes-associated protein (YAP) inhibitor (0.5 wt% NGO/GelMA + 10 μM verteporfin, Selleckchem).

Techniques: Translocation Assay

Journal: Cell Reports

Article Title: Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion

doi: 10.1016/j.celrep.2021.110227

Figure Lengend Snippet: PKN2 modulates TEAD-driven transcription and nuclear localization of the mechanosensor YAP (A) Schematic showing potential downstream targets of PKN2 involved in myofibroblast differentiation. (B–D) Normalized expression of SRF (B), TEAD (C), or SMAD (D) responsive Firefly luciferase reporter in WT and KO PSCs starved in 0.5–1% serum or treated with 5 ng/mL TGF-β1 or 10% serum. Values are normalized to a Renilla luciferase control per sample and presented relative to WT serum-starved PSCs (n = 5; two-way ANOVA with Tukey's correction). (E) qPCR analysis of expression of indicated genes in PKN2 WT and KO PSCs expressed as a fold change to WT control (n = 4). (F) Immunofluorescent images of YAP1 localization (green) in WT and PKN2 KO PSCs plated at low and high density on glass coverslips for 48 h (minimum of 100 cells/condition; n = 3; scale bar represents 50 μm). (G) Percentage of WT and PKN2 KO PSCs with YAP-positive nuclei plated at both high and low density (n = 3; unpaired t test). (H) Quantification by Python CellProfiler algorithm of YAP nuclear intensity for indicated number of cell neighbors (n = 3; two-way ANOVA with Sidak's test). (I and J) Representative western blot and quantification of p-YAP S112 and total YAP expression in WT and PKN2 KO PSCs plated at low and high density (n = 3; two-way ANOVA with Tukey's multiple comparisons test). (K and L) Western blot and quantification of p-SMAD2/3 induction with 5 ng/mL TGF-β1 for indicated time points; quantification expressed relative to untreated WT PSCs (n = 3; unpaired t test). (M) Western blot analysis of p-p70 S6K, total p70 S6K, p-ERK1/2, and total ERK in WT and PKN2 KO PSCs starved in 1% serum and treated with vehicle or 5 ng/mL TGF-β1 for 4 h (n = 2). For statistics: ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; and ∗∗∗∗ p < 0.0001.

Article Snippet: YAP WT (YAP1-V5 in pLX304) , William Hahn - Addgene; , Cat. No.: 42555; RRID:Addgene_42555; http://n2t.net/addgene:42555.

Techniques: Expressing, Luciferase, Control, Western Blot

Journal: Cell Reports

Article Title: Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion

doi: 10.1016/j.celrep.2021.110227

Figure Lengend Snippet: PKN2 loss reduces PSC-led cancer cell invasion but promotes cancer cell outgrowth. (A) Bright-field (top; scale bar represents 200 μm) and live-cell confocal z stack projections (bottom; scale bar represents 100 μm) of spheroids (n > 16) containing H2B-RFP TB32048 PDAC cells (red) and H2B-GFP WT or PKN2 KO PSCs (green) embedded in Matrigel matrix for 3 days after siRNA treatment. (B and C) Area of fibroblast-led invasion (B) or cancer cell outgrowth (C) per spheroid, normalized to total spheroid area and expressed as fold change relative to WT control (n > 16 spheroids/condition; one-way ANOVA with Tukey's multiple comparisons test). (D) Bright-field (top panel) and confocal (bottom panels) images of spheroids containing TB32048 cancer cells with WT or PKN2 KO PSCs transduced with either empty vector (EV), YAP WT (YAP), or YAP S6A (S6A) vectors. Dotted white lines indicate core area of spheroid. (E and F) Quantification of area of PSC-led (E) or epithelial (F) invasion, normalized to total spheroid area per spheroid, relative to EV (n = 3; two-way ANOVA with Tukey's multiple comparisons test). (G) Dual luciferase analysis of TEAD reporter on WT PSCs transduced with EV, YAP, or S6A YAP. Data expressed as Firefly or Renilla luminescence for each well relative to EV (n > 3; two-way ANOVA with Tukey's multiple comparisons test; ∗p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001).

Article Snippet: YAP WT (YAP1-V5 in pLX304) , William Hahn - Addgene; , Cat. No.: 42555; RRID:Addgene_42555; http://n2t.net/addgene:42555.

Techniques: Control, Transduction, Plasmid Preparation, Luciferase

Journal: Cell Reports

Article Title: Disruption of pancreatic stellate cell myofibroblast phenotype promotes pancreatic tumor invasion

doi: 10.1016/j.celrep.2021.110227

Figure Lengend Snippet:

Article Snippet: YAP WT (YAP1-V5 in pLX304) , William Hahn - Addgene; , Cat. No.: 42555; RRID:Addgene_42555; http://n2t.net/addgene:42555.

Techniques: Transduction, Virus, Plasmid Preparation, Recombinant, In Vivo, SYBR Green Assay, In Vitro, Membrane, Stripping Membranes, Luciferase, RNA Sequencing, Gene Expression, Control, Sequencing, Expressing, Derivative Assay, Software