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: Nature Communications

Article Title: Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer

doi: 10.1038/s41467-024-47423-0

Figure Lengend Snippet: a Schematic representation of YAP nuclear localization and interaction with transcription factors in DTPs. Created with BioRender.com. b , c YAP levels in nuclear lysates evaluated across PC9 and H1975 cells treated with osimertinib (Osim.), H3122 and H2228 cells treated with alectinib (Alec.), as well as H358 cells and H1838 cells treated with SHP2 inhibitor RMC-4550. Lysates were collected at indicated time points. For PC9 cells, a detailed time course is presented, including corresponding osimertinib-resistant PC9-AR. n = 3 independent experiments. d PanTEAD-YAP proximity ligation assay (PLA) in PC9 cells treated with osimertinib, analyzed by confocal microscopy. Image is representative of 200 or more cells per condition in total n = 4 independent experiments. e , f Quantification of percentage of cells from ( d ), mean ± s.d., two-sided t test, *** p = 1.08 x 10 -4 (DMSO vs. Osim.-2d), *** p = 7.32 x 10 -21 (DMSO vs. Osim.-9d), ** p = 0.0023 (DMSO vs. washout), ** p = 2.35 x 10 -43 (Osim.-9d vs. washout). g , h YAP target genes were significantly induced in PC9 and H3122 DTPs. n = 3 independent experiments, mean ± s.d., two-sided t test, ( g ) ** p = 0.0028, ( h ) ** p = 0.0018, *** p < 0.0001. i , j YAP target genes were significantly induced in DTP-specific states versus acquired-resistant states in PC9 and H3122 DTPs. n = 3 independent experiments, mean ± s.d., two-sided t test, ( i ) ** p = 0.0031, ( j ) ** p = 0.0002, ( i , j ) *** p < 0.0001. k Significant decrease in relative DTP cell numbers upon siRNA-mediated YAP knockdown during DTP development in PC9 and H3122 cells. n = 3 independent experiments, mean ± s.d., two-sided t test. l Significant decrease in relative cell numbers upon siRNA-mediated YAP knockdown in PC9 and H2228 DTPs. n = 3 independent experiments, mean ± s.d., two-sided t test. m Cell viability of PC9 DTPs was significantly decreased by treatment with TEAD inhibitor VT-104 (TEADi). n = 3 independent experiments, mean ± s.d., two-sided t test, ** p = 0.0024, ** p = 0.0023, ** p = 0.0026, *** p < 0.0001. n Compared to parental or AR cells, combined treatment with osimertinib and TEADi exerted a significant effect and reduced cell viability, specifically in PC9 DTPs. n = 3 independent experiments, mean ± s.d., two-sided t test, ** p = 0.0024, ** p = 0.0023, * p = 0.0198, *** p < 0.0001. o Gene set enrichment analysis (GSEA) for the YAP-5SA_UP gene set (Supplementary Data ) using RNAseq expression data from untreated parental control compared to PC9 DTPs, H3122 DTPs and H358 DTPs. NES, Nominal Enrichment Score; FDR, False Discovery Rate.

Article Snippet: Full-length YAP was amplified by PCR using forward primer 5’-TTTGACCTCCATAGAAGATTCTAGATGGAACAAAAACTCATCTC-3’ and reverse primer 5’-AGCGATCGCAGATCCTTCGCGGCCGCTATAACCATGTAAGAAAGCTTTC-3’ from pQCXIH expression constructs encoding myc-tagged YAP-WT (Addgene #33091), YAP-5SA (Addgene #33093), and YAP-S94A (Addgene #33094), respectively.

Techniques: Proximity Ligation Assay, Confocal Microscopy, Expressing

Journal: Nature Communications

Article Title: Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer

doi: 10.1038/s41467-024-47423-0

Figure Lengend Snippet: a Schematic representation of the establishment and treatment study of the humanized EGFR-mutant PC9 xenograft model. A schematic diagram was created with BioRender.com. b Changes in tumor volume at treatment endpoint. A Osimertinib [5 mg/kg] treatment study in the humanized PC9 mouse model was conducted comparing parental cells, cells expressing YAP-WT and cells expressing hyperactive YAP-5SA. n = 3 mice, mean ± SEM, two-sided t test. c – e Changes in tumor-infiltrating macrophage populations at treatment endpoint. Macrophage populations are defined as CD11b+ cells, with HLA-DR+ for M1 macrophages and CD163+ for M2 macrophages. n = 3 mice, mean ± SEM, two-sided t test. ( c ) The sequential gating strategies are provided in Supplementary Fig. . f – i Changes in tumor infiltrating T cell populations at treatment endpoint. T-cell populations are defined as CD25+/CD3+ cells, with differentiation of CD4+ and cytotoxic CD8+ T-cells. f The sequential gating strategies are provided in Supplementary Fig. . n = 3 mice, mean ± SEM, two-sided t test.

Article Snippet: Full-length YAP was amplified by PCR using forward primer 5’-TTTGACCTCCATAGAAGATTCTAGATGGAACAAAAACTCATCTC-3’ and reverse primer 5’-AGCGATCGCAGATCCTTCGCGGCCGCTATAACCATGTAAGAAAGCTTTC-3’ from pQCXIH expression constructs encoding myc-tagged YAP-WT (Addgene #33091), YAP-5SA (Addgene #33093), and YAP-S94A (Addgene #33094), respectively.

Techniques: Mutagenesis, Expressing

Journal: Nature Communications

Article Title: Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer

doi: 10.1038/s41467-024-47423-0

Figure Lengend Snippet: a Normalized expression of YAP signature genes across patient specimen classified as TN, RD or progressive disease (PD) using previously published single cell RNA-seq (scRNA-seq) data 1 . The YAP signature determined by genes within the YAP-5SA_UP gene set that are significantly upregulated across PC9, H3122, and H358 DTPs, and were also differentially upregulated in the RD treatment timepoint. The sequencing data were filtered to limit analyzes to malignant lung epithelial cells only (N cells: TN = 621, RD = 484, PD = 138). P-values obtained from two-sided Dunn’s test with Bonferroni adjustment. The box plot displays 25th (lower bound), 50th (centre, median), and 75th (upper bound) percentiles, with whiskers (minima (bottom), maxima (top)) extending 1.5 * IQR. b Average expression of individual YAP signature genes highlighted across TN, RD, and PD treatment groups. c Immunohistochemistry staining for YAP in patient specimens classified as TKI treatment naïve (TN) or collected at residual disease upon treatment with targeted inhibitors (RD). Quantification of nuclear levels (% nuclear) by automated image analysis. Arrows indicate YAP-positive tumor cell nuclei, scale bar: 20 μm. Statistical analysis by two-sided t-test. *** p = 0.0007, n = 18 independent experiments. d Single-cell RNAseq analysis of clinical samples showed enrichment of the FAK signature in the residual disease state. The significant FAK features include NEDD9, PTPRE, MAP1B, PTRF and NOV. Violin plot data points are single cells’ mean expression of FAK signature. P-values obtained from two-sided Dunn’s test with Bonferroni adjustment.

Article Snippet: Full-length YAP was amplified by PCR using forward primer 5’-TTTGACCTCCATAGAAGATTCTAGATGGAACAAAAACTCATCTC-3’ and reverse primer 5’-AGCGATCGCAGATCCTTCGCGGCCGCTATAACCATGTAAGAAAGCTTTC-3’ from pQCXIH expression constructs encoding myc-tagged YAP-WT (Addgene #33091), YAP-5SA (Addgene #33093), and YAP-S94A (Addgene #33094), respectively.

Techniques: Expressing, RNA Sequencing Assay, Sequencing, Immunohistochemistry, Staining