Journal: Clinical Cancer Research

Article Title: Targeting Neuropilin 1 as an Antitumor Strategy in Lung Cancer

doi: 10.1158/1078-0432.ccr-07-0001

Figure Lengend Snippet: Fig. 5. Cyclic 7-mer peptides bind NRP1and inhibit CL1-5 invasion and angiogenesis in vivo. A, the selected peptides reduce phosphorylation ofVEGFR2. HUVECs were pretreated with peptides for10 min followed by treatment withVEGF for 5 min. Phosphorylation ofVEGFR2 was determined byWestern blotting with an anti ^ phospho-VEGFR2 antibody.TotalVEGFR2 was determined byWestern blotting with an anti-VEGFR2 antibody. B, the effect of peptides on CL1-5 cells invasive activity. The invasive activity of cells was detected by invasion assay. CL1-5 cells (2.5 104) were seeded onTranswells coated with 30 Ag matrigel and incubated with peptides DG1or DG2 for 48 h.Then, the cells that had invaded the membrane were counted.Values were normalized to the relative invasion activity of the nontreated control cells. Experiments were done in triplicate, three independent times. In DG1- and DG2-treated cells, the invasion ability was statistically significantly different across the various concentrations of DG1or DG2 byANOVA (DG1, P < 0.001; DG2, P = 0.011). C, the effect of peptides on tumor angiogenesis in vivo. Immunohistochemical staining of the Matrigel plug sections with an anti-CD31antibody showed a significant decrease in CD31-positive vessels in plugs containing DG1peptide compared with mock-treated plugs. Original magnification, 200.The counts of microvessels surrounding the tumor nests were calculated. D, effect of peptides on tumorigenesis in vivo.Volumes of tumors from control CL1-5 cells (E) and DG1-treated cells (n) were measured at the indicated times as described in Materials and Methods. Means and 95% CI are shown (n = 5 mice per group). E, summary diagram showing thatVEGF165 can bind to NRP1and trigger the NRP1/VEGFR2/PI3K/Akt signaling pathways and result in tumor angiogenesis, cancer cell invasion, and tumorigenesis.The synthetic peptides DG1/DG2 can specifically block this signaling pathway and may have therapeutic potential.

Article Snippet: Human umbilical vascular endothelial cells (HUVEC) and culture media were purchased from Cell Applications, Inc.

Techniques: In Vivo, Phospho-proteomics, Activity Assay, Invasion Assay, Incubation, Membrane, Control, Immunohistochemical staining, Staining, Protein-Protein interactions, Blocking Assay

Journal: Nature communications

Article Title: Stress-induced RNA-chromatin interactions promote endothelial dysfunction.

doi: 10.1038/s41467-020-18957-w

Figure Lengend Snippet: Fig. 1 High glucose and TNFα induce a profound gene expression and phenotypic change. a HUVECs were treated in biological duplicates as Day 0: 25 mM mannitol as normal glucose and osmolarity control (NM); Day 3: combined treatment consisting 25 mM D-glucose and 5 ng/mL TNFα (H + T) for 3 days; and Day 7: H + T treatment for 7 days. Each group of treated cells was subjected to single-cell RNA-seq (scRNA-seq), Hi-C, and iMARGI assays. b t-SNE plot of scRNA-seq (4000–15,000 cells per sample) showed clear separation by treatment condition into three distinct clusters. c Principal component analysis of scRNA-seq data: single cells are plotted in the first two PC space and are labeled in red (Day 0, i.e., NM), green (Day 3, i.e., 3-day H + T treatment), and blue (Day 7, i.e., 7-day H + T treatment). d Expression heatmap (z-scaled) of top DE genes in single ECs grouped into functional pathways. Cells were ordered by increasing SERPINE1 expression (per each sample separately) and binned per 100 cells for the analysis. A total of 269 bins in Day 0, 177 bins in Day 3, and 148 bins in Day 7. e t-SNE plots of the expression level of selected genes in each single cell across the time course. The RNA levels are represented by log-normalized unique molecular identifier counts. f mRNA levels of eNOS and α-SMA in NM vs. H + T-treated HUVECs and cells untreated (NT) or treated with TGF-β (10 ng/mL) and IL-1β (5 ng/mL; T + I) for 3 or 7 days. The respective control was set as 1. Relative eNOS level: data represent mean ± SEM from five independent experiments; relative α-SMA level in H + T treatment: data represent mean ± SEM from seven independent experiments; relative α-SMA level in T + I treatment: data represent mean ± SEM from four independent experiments. *P = 0.0067, 0.0087, 0.0057, and 0.0017 from left to right based on ANOVA with Bonferroni as post hoc test. g Cell morphology under bright field (BF), immunofluorescent staining of α-SMA, and VE-cadherin (VE-cad), phalloidin staining of cytoskeleton, and DRAQ5 (DRAQ) staining of the nuclei. Representative images from five independent experiments are shown. Scale bar of BF = 100 µm; scale bars of (immuno)fluorescent staining = 50 µm. Source data are provided as a Source data file.

Article Snippet: Plasmid transfection was performed using the CytofectTM HUVEC Transfection kit (Cell Applications) following the manufacturer’s protocol in 6-well or 12-well plates.

Techniques: Gene Expression, Control, RNA Sequencing, Hi-C, Labeling, Expressing, Functional Assay, Staining

Journal: Nature communications

Article Title: Stress-induced RNA-chromatin interactions promote endothelial dysfunction.

doi: 10.1038/s41467-020-18957-w

Figure Lengend Snippet: Fig. 2 Overview of time-course Hi-C and iMARGI data. a, b Proportions of intrachromosomal (yellow) and interchromosomal read pairs (blue) in Hi-C (a) and iMARGI data (b) at the three time points (columns). c An example of interchromosomal iMARGI read pairs mapped to chromosome 2 near LINC00607 (left) and chromosome 7 near SERPINE1 (right). The RNA end (pink) and the DNA end (green) of each read pair is linked by a horizontal line. d Examples of overlapping iMARGI read pairs on a contact matrix from the RNA end (rows) to the DNA end (columns) with SEs (marked in light blue and in SE tracks) in control (Day 0) ECs. Genome region: chr1:75,000,0000–chr1:125,000,000. Resolution = 200 kb. e Proportions of iMARGI read pairs with the RNA ends (pink) or the DNA ends (green) in Day 0 (Ctrl) and Days 3 and 7 (H + T) ECs mapped to HUVEC SEs. Dotted line: the relative size of HUVEC SEs compared to the size of the genome. Source data are provided as a Source data file.

Article Snippet: Plasmid transfection was performed using the CytofectTM HUVEC Transfection kit (Cell Applications) following the manufacturer’s protocol in 6-well or 12-well plates.

Techniques: Hi-C, Control

Journal: Nature communications

Article Title: Stress-induced RNA-chromatin interactions promote endothelial dysfunction.

doi: 10.1038/s41467-020-18957-w

Figure Lengend Snippet: Fig. 4 Inhibition of LINC00607 attenuates SERPINE1 induction and EC dysfunction. a Illustration of LINC00607 genomic locus and gene structure and LNA GapmeRs targeting of LINC00607 RNA. b HUVECs were transfected with scramble (scr) or two LNAs targeting LINC00607. LINC00607 RNA and SERPINE1 mRNA levels were quantified. Data represent mean ± SEM from seven independent experiments. c qPCR of LINC00607 in subcellular fractionations of HUVECs transfected with scr, LNA1, or LNA2. Data represent mean ± SEM from four independent experiments. d RNA-seq was performed with cells transfected as in b in biological replicates. Heatmap is plotted based on z-scaled log-transformed gene expression levels. e, f ECs transfected with scr or LNA1 and then treated by H + T were used in e monocyte adhesion assay and in f SA-β-gal assay. In e, the number of attached monocytes to ECs were quantified. Data represent mean ± SEM from eight independent experiments performed with peripheral blood-derived monocytes from four individual donors. Representative images show the attachment of fluorescently labeled peripheral blood-derived monocytes to ECs for experiments performed with monocytes from three different donors. In f, ECs with positive β-gal staining were quantified. The positively stained cell number in scr control was set to 100 (%). f Data represent mean ± SEM from four independent experiments respectively. Scale bar = 200 μm. * denotes p = 0.0289, 0.0013, 0.001, 0.0001, 0.0025, and 0.024 from left to right (in b) and p = 0.0023, 0.0079, and 0.0249 from left to right (in c) between indicated groups based on ANOVA followed by Bonferroni post hoc test (in b and c); p = 0.0039 (in e) and 0.0014 (in f), as compared to scr group based on two-tailed paired t test (in e and f). Source data are provided as a Source data file.

Article Snippet: Plasmid transfection was performed using the CytofectTM HUVEC Transfection kit (Cell Applications) following the manufacturer’s protocol in 6-well or 12-well plates.

Techniques: Inhibition, Transfection, RNA Sequencing, Transformation Assay, Gene Expression, Cell Adhesion Assay, Derivative Assay, Labeling, Staining, Control, Two Tailed Test