Journal: Thoracic Cancer

Article Title: Circ_0001667 accelerates breast cancer proliferation and angiogenesis through regulating CXCL10 expression by sponging miR ‐6838‐5p

doi: 10.1111/1759-7714.14820

Figure Lengend Snippet: Knockdown of circ_0001667 inhibited proliferation and angiogenesis in breast cancer cells. MDA‐MB‐231 and BT549 cells were transfected with si‐NC or si‐circ_0001667. (a) qRT‐PCR assay for silencing efficiency of si‐circ_0001667. (b‐d) CCK8 and EdU assays to detect proliferation of breast cancer cells. Scale bar: 50 μm. (e and f) Flow cytometry was used to determine the cycle distribution of breast cancer cells. (g) Colony formation assay was used to assess the colony formation ability. (h) Western blot to measure the protein levels of PCNA and Ki67. (i) Tube formation assay was used to analyze angiogenesis of breast cancer cells. (j) Western blot showing the expression levels of VEGFA and FGF2 in breast cancer cells. * p < 0.05.

Article Snippet: The separated proteins were electrotransferred to PVDF membrane (Beyotime), closed with 10% skimmed milk, and incubated with primary antibodies (Abcam) against PCNA (ab18197, 1:1000), Ki67 (ab254123, 1:5000), VEGFA (ab46154, 1:2000), FGF2 (ab208687, 1:1000), CXCL10 (ab9807, 1:2000), or β‐actin (ab6276, 1:5000).

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Flow Cytometry, Colony Assay, Western Blot, Tube Formation Assay, Expressing

Journal: Thoracic Cancer

Article Title: Circ_0001667 accelerates breast cancer proliferation and angiogenesis through regulating CXCL10 expression by sponging miR ‐6838‐5p

doi: 10.1111/1759-7714.14820

Figure Lengend Snippet: Downregulation of miR‐6838‐5p attenuated the effect of circ_0001667 knockdown on cell proliferation and angiogenesis. (a) qRT‐PCR analysis of miR‐6838‐5p levels in cells transfect with anti‐miR‐6838‐5p or anti‐miR‐NC. (b‐j) MDA‐MB‐231 and BT549 cells were transfected with si‐NC, si‐circ_0001667, si‐circ_0001667 + anti‐miR‐NC or si‐circ_0001667 + anti‐miR‐6838‐5p. (b‐d) CCK‐8 and EdU assays detected cell proliferation. (e and f) Flow cytometry determined cell cycle distribution. (g) The colony formation assay assessed the colony formation ability. (h) Western blot measured the protein levels of PCNA and Ki67. (i) Tube formation test analyzed angiogenesis. (j) Western blot showed the expression levels of VEGFA and FGF2. * p < 0.05.

Article Snippet: The separated proteins were electrotransferred to PVDF membrane (Beyotime), closed with 10% skimmed milk, and incubated with primary antibodies (Abcam) against PCNA (ab18197, 1:1000), Ki67 (ab254123, 1:5000), VEGFA (ab46154, 1:2000), FGF2 (ab208687, 1:1000), CXCL10 (ab9807, 1:2000), or β‐actin (ab6276, 1:5000).

Techniques: Knockdown, Quantitative RT-PCR, Transfection, CCK-8 Assay, Flow Cytometry, Colony Assay, Western Blot, Expressing

Journal: Thoracic Cancer

Article Title: Circ_0001667 accelerates breast cancer proliferation and angiogenesis through regulating CXCL10 expression by sponging miR ‐6838‐5p

doi: 10.1111/1759-7714.14820

Figure Lengend Snippet: Overexpression of CXCL10 reversed the effects of miR‐6838‐5p overexpression on cell proliferation and angiogenesis. (a and b) Validation of CXCL10 overexpression by qRT‐PCR and western blot. (c–k) MDA‐MB‐231 and BT549 cells were transfected with miR‐NC, miR‐6838‐5p, miR‐6838‐5p + pcDNA or miR‐6838‐5p + CXCL10. (c–h) CCK8 assay, EdU assay, flow cytometry and colony formation assay detected cell proliferation. (i) Western blot analysis for PCNA and Ki67 levels. (j) Tube formation test detected angiogenesis. (k) Western blotting quantified the levels of VEGFA and FGF2. * p < 0.05.

Article Snippet: The separated proteins were electrotransferred to PVDF membrane (Beyotime), closed with 10% skimmed milk, and incubated with primary antibodies (Abcam) against PCNA (ab18197, 1:1000), Ki67 (ab254123, 1:5000), VEGFA (ab46154, 1:2000), FGF2 (ab208687, 1:1000), CXCL10 (ab9807, 1:2000), or β‐actin (ab6276, 1:5000).

Techniques: Over Expression, Biomarker Discovery, Quantitative RT-PCR, Western Blot, Transfection, CCK-8 Assay, EdU Assay, Flow Cytometry, Colony Assay

Journal: Diagnostics

Article Title: Development of a Flow-Free Automated Colorimetric Detection Assay Integrated with Smartphone for Zika NS1

doi: 10.3390/diagnostics10010042

Figure Lengend Snippet: ( a ) Sandwich ELISA assay results using the recombinant Zika nonstructural protein 1 (NS1) antigen spiked on PBS buffer on 96-well plate as the Zika NS1 monoclonal antibody is used as a capture agent. Anti-Zika NS1 monoclonal antibody-HRP with a dilution factor of 1:1000 utilized to react with TMB calorigenic substrate to develop color. ( b ) M-ELISA assay on a 96-well plate showing spiking recombinant Zika NS1 on whole plasma. A 1:500 dilution factor of HRP-labeled anti-Zika NS1 was used and reacted with TMB to generate color. In both cases, color development was stopped by using H 2 SO 4 and absorbance was measured at 450 nm using a SpectraMax Gemini™ XPS/EM Microplate Reader (Molecular Devices, USA). Error bars are ±SD.

Article Snippet: It was rewashed three times carefully, followed by 90 min incubation of 100 μL of recombinant Zika NS1 antigen (BF-NS1-6309, Biofront Technologies, Tallahassee, Florida, USA) with different concentrations spiked in PBS at room temperature.

Techniques: Sandwich ELISA, Recombinant, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Labeling

Journal: bioRxiv

Article Title: SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

doi: 10.1101/2021.12.10.472112

Figure Lengend Snippet: (A) Schematic depicting S-triggered barrier dysfunction measured by a trans-endothelial/epithelial electrical resistance assay (TEER; left) and an endo/epithelial glycocalyx layer (EGL) assay (right). (B) Time course TEER assay measuring the barrier function of HPMEC monolayers over time with the indicated treatments, including DENV2 NS1 (5 μg/mL), VEGF (50 ng/mL), and SARS-CoV-2 S (10 μg/mL). Data are from n=2 biological replicates. (C) A TEER assay measuring the barrier of monolayers of HPMEC and HPMEC/ACE2 at 24 hours after the indicated treatments. VEGF positive control (50 ng/mL). Data are from n=3 biological replicates. (D) Same as C but treated with the indicated VSV pseudotyped particles at the indicated dilutions. VSV-bald and VSV-G are diluted 1:30. Data are from n=3 biological replicates. (E) Same as C but treated with the indicated concentrations of SARS-CoV-2 RBD. Data are from n=3 biological replicates. (F) Same as C but measuring the barrier of Calu-3 cell monolayers. Data are from n=2 biological replicates. (G) A TEER inhibition assay measuring the capacity of a cocktail of anti-S antibodies to inhibit S-mediated endothelial hyperpermeability. S (10 μg/mL) and the antibody cocktail (15 μg/mL for each antibody; 1A9 [Genetex] and CR3022 [Absolute Antibody]) were added simultaneously to the upper chamber of transwell inserts to a monolayer of HPMEC or HPMEC/ACE2 and TEER was measured 24 hours post-treatment (hpt). Data are from n=2 biological replicates. In all panels, the dotted line is the normalized TEER value of the untreated control condition. All data are plotted as mean +/− SD. For all panels, values are compared to untreated controls by ANOVA with multiple comparisons with *p<0.05, **p<0.01, ***p<0.001, and n.s. p>0.05.

Article Snippet: Purified proteins were formulated at ~1 mg/mL in PBS and stored in aliquots at −80° C. Recombinant DENV serotype 2 NS1 was purchased from the Native Antigen Company and characterized previously (Dengue virus serotype 2 NS1 [accession # P29990.1, Thailand/16681/84]) ( ).

Techniques: Positive Control, Inhibition

Journal: bioRxiv

Article Title: SARS-CoV-2 Spike triggers barrier dysfunction and vascular leak via integrins and TGF-β signaling

doi: 10.1101/2021.12.10.472112

Figure Lengend Snippet: (A) A representative mouse back from a dermal leak experiment. The dorsal dermises of mice were injected intradermally with the treatments and doses indicated. Mice then received a dextran-680 tracer molecule intravenously. Mouse dermises were collected 2 h post-treatment and quantification of local dermal leak was assessed by a fluorescent scanner. (B) Quantification of A from mice treated with PBS (n=27), DENV2 NS1 (15 μg; n=5), S (10 μg; n=25), and S (25 μg; n=5). (C) Representative lung images from a SARS-CoV-2 S systemic vascular leak assay. Mice were administered 50 μg of SARS-CoV-2 S or ovalbumin intranasally as indicated, and 22 hpt were administered a dextran-680 tracer intravenously as in A. Organs of mice were collected 2 hours post dextran-680 administration (24 hours post-S treatment), and accumulation of dextran-680 was measured with a fluorescent scanner. (D) Quantification of C from n=6 mice. (E) Same as C except representative images of spleens. (F) Quantification of E from n=6 mice. (G) Same as C except representative images of small intestine. (H) Quantification of G from n=5 mice. MFI is mean fluorescence intensity. All data are plotted as mean +/− SEM with *p<0.05, **p<0.01, and ***p<0.001 by unpaired t-test.

Article Snippet: Purified proteins were formulated at ~1 mg/mL in PBS and stored in aliquots at −80° C. Recombinant DENV serotype 2 NS1 was purchased from the Native Antigen Company and characterized previously (Dengue virus serotype 2 NS1 [accession # P29990.1, Thailand/16681/84]) ( ).

Techniques: Injection, Fluorescence

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: (A-E) Human endothelial cells from different tissues (A, lung; B, skin; C, umbilical vein; D, brain; and E, liver) were grown on Transwell semi-permeable membranes (0.4 μm pore size), and distinct flavivirus NS1 proteins (5 μg/mL, 1.5 μg total protein) were added to the apical chamber. TNF-α (10 ng/mL) was used as a positive control, and DENV2 recombinant envelope (E) protein (5 μg/mL) was used as a negative control. A TEER assay was used to evaluate the effect of these NS1 proteins on endothelial permeability at indicated time-points over 48 h. ^ represents media change. Relative TEER values from two independent experiments performed in duplicate are plotted. Error bars indicate SEM. Statistically significant differences between distinct groups (all time points combined for each group) compared to the untreated groups were determined by a two-way ANOVA analysis using Dunnett’s test for multiple comparisons, with ****p < 0.0001 and ns (not significant), p > 0.05.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Pore Size, Positive Control, Recombinant, Negative Control, Permeability

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: Binding of flavivirus NS1 proteins to the surface of different human endothelial cell monolayers as measured by (A-E) confocal microscopy or (F-J) western blot. (A-E) (A) HPMECs, (B) HMEC-1, (C) HUVECs, (D) HBMECs, and (E) HLSECs were grown on coverslips and treated with 5 μg/mL NS1 or10 μg/mL NS1 (1.5 μg or 3 μg of total protein, respectively) as indicated (also see ); binding was evaluated 1 h post-treatment (hpt). The amount of bound NS1 was quantified and expressed as MFI. (F-J) Cell lysates from (F) HPMECs, (G) HMEC-1, (H) HUVECs, (I) HBMECs, and (J) HLSECs treated with 10 μg/mL (3 μg total protein) of flavivirus NS1 protein and collected 1 hpt and analyzed by western blot . All graphs show the average of quantification from two independent experiments run in duplicate. Error bars indicate SEM. In (A)-(E), statistically significant differences between distinct treatment groups compared to the untreated group [NS1(−)] were determined by a two-way ANOVA analysis using Dunnett’s test for multiple comparisons, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Binding Assay, Confocal Microscopy, Western Blot

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: (A) Sialic acid expression on human endothelial cell monolayers grown on coverslips 6 hpt with different flavivirus NS1 proteins (5 μg/mL, 1–5 μg total protein), examined by confocal microscopy. Sialic acid was stained with wheat germ agglutinin conjugated to Alexa Fluor 647 (red). Nuclei were stained with Hoechst (blue). Images (20×) are representative of three independent experiments. Scale bar, 10 μm. (B) Quantification of MFI in (A) from three independent experiments. Error bars indicate SEM. Reduction of sialic acid expression in NS1-treated monolayers was normalized to untreated controls (dotted line). Statistically significant differences between distinct treatment groups compared to the untreated group were determined by a two-way ANOVA analysis using Dunnett’s test for multiple comparisons, with *p < 0.05, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Expressing, Confocal Microscopy, Staining

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: (A) Heparan sulfate expression on human endothelial cell monolayers grown on coverslips 6 hpt with different flavivirus NS1 proteins (5 μg/mL, 1–5 μg total protein), examined by confocal microscopy. Heparan sulfate was stained with anti-heparan sulfate mAb (green). Nuclei were stained with Hoechst (blue). Images (20×) are representative of three independent experiments. Scale bar, 10 μm. (B) Quantification of MFI in (A) from three independent experiments. Error bars indicate SEM. Reduction of heparan sulfate expression in NS1-treated monolayers was normalized against untreated controls (dotted line). Statistically significant differences between distinct treatment groups compared to the untreated group were determined by a two-way ANOVA analysis using Dunnett’s test for multiple comparisons, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Expressing, Confocal Microscopy, Staining

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: (A and B) Fold change over untreated controls (dotted lines) of the quantification of MFI from three independent confocal microscopy experiments examining (A) expression and (B) activity of cathepsin L (Magic Red reagent) in human endothelial cell monolayers 6 hpt with different flavivirus NS1 proteins (5 μg/mL, 1–5 μg total protein) . (C) Western blot data of pro- and mature- cathepsin L expression in human endothelial cell monolayers 6 hpt with different flavivirus NS1 proteins (5 μg/mL, 1.5 μg total protein). (D and E) Densitometry of bands from (C) for (D) pro-cathepsin L and (E) mature cathepsin L with values normalized to GAPDH and expressed as fold change from untreated monolayers. (F) Fold change over untreated controls (dotted line) of the quantification of MFI from three independent confocal microscopy experiments examining the expression of heparanase in human endothelial cell monolayers 6 hpt with different flavivirus NS1 proteins (5 μg/mL, 1.5 μg total protein) . For all graphs, error bars indicate SEM. Statistically significant differences between distinct treatment groups compared to the untreated group were determined by a two-way ANOVA analysis using Dunnett’s test for multiple comparisons, with *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Confocal Microscopy, Expressing, Activity Assay, Western Blot

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: (A-H) Wild-type C57BL/6 mice were used to evaluate (A and B) local or (C-H) systemic vascular leakage induced by flavivirus NS1 proteins injected either (A and B) intradermally (dorsal dermis) or (C-H) intravenously with 15 μg and 200 μg (10 mg/kg) of total protein, respectively. The magnitude of the vascular leakage induced by these proteins was evaluated by using (A, B, and E-H) Alexa-Fluor-680-conjugated dextran (10 mg/mL) or (C and D) Evans blue dye (0.5%) and quantified by (A, B, and E-H) fluorescent imaging or (C and D) spectrophotometric analyses. (A, E, and F) Images (LI-COR Odyssey) and (B-D, G, and H) MFI values are representative of two individual experiments (n = 3–6 per group). Ovalbumin (OVA; 10 mg/kg) and PBS were used as controls for systemic and local vascular leakage experiments, respectively. (I) Levels of heparan sulfate detected in serum of mice (n = 3 per group) treated with ovalbumin (OVA), DENV2 NS1, WNV NS1, or YFV NS1 (all 10 mg/kg) as measured by ELISA. Untreated mice were used as a steady-state control. For dextran-adapted dermal Miles assay, data were derived from three independent experiments and expressed as the fold-change ratio of the MFI obtained in each individual treatment group compared to PBS injection (dotted line in B). MFI was measured inside the leakage area by drawing a standard circular area in each treatment condition using ImageJ tools. Statistically significant differences between distinct treatment groups compared to the control groups were determined by a one-way ANOVA analysis using Dunnett’s test for multiple comparisons, with *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns (not significant), p > 0.05 . Error bars indicate SEM. Scale bars represent (A) 2.5 cm and (E and F) 0.5 cm.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Injection, Imaging, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet: Flavivirus infections can affect specific tissues, including brain, lung, skin, liver, and the placenta during pregnancy, to cause neurotropic-encephalitic or systemic diseases in humans. Infection with any of the flaviviruses leads to secretion of NS1, a soluble viral protein that directly triggers endothelial hyperpermeability and vascular leakage associated with the disruption of key components of the endothelial glycocalyx layer, which lines the luminal surface of endothelial cells in the vasculature. The EGL is composed of monosaccharides such as sialic acid (Sia), glycosaminoglycans (GAGs) such as heparan sulfate, and heparan sulfate proteoglycans (HSPGs) such as syndecan-1. Here, we describe an endothelial cell-type-dependent increase in permeability and vascular leakage induced by different flavivirus NS1 proteins that reflect the disease pathogenesis of distinct flavivirus infections. DENV NS1 increases the permeability of endothelial cells from multiple tissues, such as lung, skin, umbilical vein, brain, and liver, consistent with the systemic disease caused by DENV. ZIKV NS1 induces endothelial dysfunction of umbilical vein and brain endothelial cells, reflecting severe congenital and neurological defects associated with Zika, while WNV and JEV NS1 only affect the barrier function of brain endothelial cells, in line with the neurotropic and encephalitic nature of WNV and JEV disease. Finally, YFV NS1 increases the permeability of lung and especially liver endothelial cells, consistent with hepatic and systemic pathology of yellow fever virus. This selective permeability may contribute to the pathogenesis of the different flaviviruses by either inducing extravasation of fluids that result in inflammation of tissues or facilitating virus dissemination into target organs that may lead to enhanced viral infection and disease.

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Infection, Disruption, Permeability, Virus

Journal: Cell reports

Article Title: Flavivirus NS1 Triggers Tissue-Specific Vascular Endothelial Dysfunction Reflecting Disease Tropism

doi: 10.1016/j.celrep.2019.01.036

Figure Lengend Snippet:

Article Snippet: Recombinant West Nile virus NS1 (NY99) protein produced in 293 human cells. C-terminal 6x His-Tag , The Native Antigen Company , NA.

Techniques: Purification, Marker, Recombinant, Virus, Produced, Membrane, Antibody Labeling, Cell Culture, Plasmid Preparation, Software