Journal: Nature cardiovascular research

Article Title: SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary vessels

doi: 10.1038/s44161-023-00336-5

Figure Lengend Snippet: a) Representative images of AI-based RNA-FISH quantification showing NRP1 RNA, SARS-CoV-2 S and S antisense+ cells. Scale bars, 50 μm. Quantification of the frequency of SARS-CoV-2 S+ and SARS-CoV-2 S antisense+ cells in macrophages and foam cells treated with non-targeting siRNA control or siRNA NRP1 at 24 hpi. Data are presented as mean values ± s.e.m. Statistical analysis was performed using unpaired two-tailed Student’s t-test. b) Representative images of RNA-FISH quantification showing NRP1 RNA, SARS-CoV-2 S and S antisense+ cells. Scale bars, 20 μm. Quantification of the frequency of SARS-CoV-2 S+ and SARS-CoV-2 S antisense+ cells in macrophages and foam cells with and without NRP1 inhibition (EG00229 trifluoroacetate) at 24 hpi. c) Heat map of differentially secreted cytokine and chemokine levels from SARS-CoV-2-infected macrophages and foam cells (n = 4–5) after treatment with siRNA control or siRNA NRP1 at 24 hpi. Adjusted P values < 0.05 were considered significant. Asterisk indicates an adjusted P value < 0.05 for the comparison of SARS-CoV-2 infected and treated with NRP1 siRNA versus infected and siRNA control. Asterisks in parentheses indicate nominal P < 0.05 for the comparison between macrophages versus foam cells, *P < 0.05; **P < 0.01; ***P < 0.001. d) Heat map of differentially secreted cytokine and chemokine levels from SARS-CoV-2-infected macrophages and foam cells after NRP1 blocking (EG00229 trifluoroacetate). Results are shown as log2FC between infected and non-treated conditions. Adjusted P values < 0.05 were considered significant. Asterisk indicates an adjusted P value < 0.05 for the comparison of SARS-CoV-2 infected and treated versus infected and vehicle-treated conditions. Asterisks in parentheses indicate nominal P value < 0.05 for the comparison between macrophages versus foam cells, *P < 0.05; **P < 0.01; ***P < 0.001. e) Plot showing the relative expression of secreted cytokines and chemokines from NRP1 blocking (EG00229 trifluoroacetate) treated versus untreated SARS-CoV-2-infected atherosclerotic plaques at 48 hpi. Relative expression is represented in log2FC colored scale. Circles represent statistically significant results, and non-significant changes are represented as diamonds.

Article Snippet: Macrophages and foam cells were treated with the EG 00229 trifluoroacetate (Tocris Cat. #6986) at 100 μM final concentration for 1 hour prior to infection.

Techniques: Control, Two Tailed Test, Inhibition, Infection, Comparison, Blocking Assay, Expressing

Journal: Nature cardiovascular research

Article Title: SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary vessels

doi: 10.1038/s44161-023-00336-5

Figure Lengend Snippet: a) Dot plot showing the relative expression levels of NRP1 RNA normalized by GAPDH RNA expression in macrophages and foam cells. Average percentage of NRP1 silencing efficacy were calculated and depicted at the top (n = 4 biological replicates measured by technical duplicate per cell type, condition). b) Representative image of capillary western blot (Wes) was performed to evaluate the protein expression levels of NRP1 after siRNA NRP1 or siRNA control treatment. Target protein NRP1 (130–140 kD) and β-actin loading control blots (42 kD) are shown. c) Total NRP1 RNA copies were quantified in not-infected macrophages and foam cells treated with either siRNA control or siRNA NRP1 (n = 31 images of macrophages siRNA control; n = 26 macrophages siRNA NRP1, n = 24 foam cells siRNA control, n = 25 foam cells siRNA NRP1) at 24 hpi. d) Representative images and quantification of RNA-FISH showing NRP1 RNA in not-infected macrophages and foam cells. e) Representative images of RNA-FISH showing SARS-CoV-2 spike vRNA and NRP1 RNA (left), SARS-CoV-2 spike antisense vRNA and NRP1 RNA (right) in infected macrophages and foam cells treated with non-targeting siRNA control or siRNA NRP1 at 24 hpi. f ) Representative images of RNA-FISH showing SARS-CoV-2 S vRNA and NRP1 RNA (left), SARS-CoV-2 S antisense RNA and NRP1 RNA (right) in infected macrophages and foam cells with and without NRP1-blocking (EG00229 trifluoroacetate) at 24 hpi.

Article Snippet: Macrophages and foam cells were treated with the EG 00229 trifluoroacetate (Tocris Cat. #6986) at 100 μM final concentration for 1 hour prior to infection.

Techniques: Expressing, RNA Expression, Western Blot, Control, Infection, Blocking Assay

Journal: Nature cardiovascular research

Article Title: SARS-CoV-2 infection triggers pro-atherogenic inflammatory responses in human coronary vessels

doi: 10.1038/s44161-023-00336-5

Figure Lengend Snippet: a) Heat map of standardized z-scored gene expression of cytokines and chemokines in not-infected, SARS-CoV-2 infected macrophages and foam cells treated with non-targeting siRNA control or siRNA NRP1 at 24 hpi. b) Quantification of TGF-β1 concentration (pg mL−1) in culture supernatants of not infected (n = 4) or SARS-CoV-2 infected macrophages and foam cells with (n = 4) or without (n = 8) NRP1-blocking treatment (EG00229 trifluoroacetate) at 24 hpi. Data are presented as mean values ±s.e.m. One-way ANOVA with post-hoc Tukey’s test for multiple comparisons was performed.

Article Snippet: Macrophages and foam cells were treated with the EG 00229 trifluoroacetate (Tocris Cat. #6986) at 100 μM final concentration for 1 hour prior to infection.

Techniques: Blocking Assay, Gene Expression, Infection, Control, Concentration Assay

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Distribution of EGF and EGFR and phosphorylation status of EGFR in EGF treated cells. ( A ) ICC for EGF and EGFR in untreated cells and cells treated with EGF for 30 min Confocal imaging was done on laser scan confocal Nikon Ti microscope at 100x magnification; staining was done for nuclei (Hoechst 33342), EGF (ab9595 Abcam, Cambridge, United Kingdom) and EGFR (antibody recognizing extracellular domain of human EGFR: epitope 6-273 aa, antibody clone sc-101; Santa Cruz Biotechnology, TX, United States). The scale bars are 50 μm. ( B ) Phosphorylation of EGFR upon treatment with EGF was determined by Western blot. Whole cell lysates were obtained from cells without treatment or 30 min after treatment with recombinant EGF. Three independent experiments were done and imaged using Li-Cor system; representative Western blots are shown. Normalization was done using α-Tubulin as a loading control (Catalog number 926-42213 Li-Cor, NE, United States). Antibodies for phosphorylated EGFR included Y1068 (Catalog number #3777, Cell Signaling, MA, United States), Y1086 (Catalog number #2220S, Cell Signaling, MA, United States) and Y1173 (Catalog number ab32578 Abcam, Cambridge, United Kingdom). ( C ) Western blot quantification was done using software provided by Li-Cor and plotted using Graph Pad Prizm. This bar graph represents relative quantification of phosphorylation of EGFR (for tyrosine positions Y1068, Y1086 and Y1173) for wild type ME180 cells and mutant clone VII11 at 30 min after EGF treatment. To show relative phosphorylation values, the highest phosphorylation signal (that for Tyrosine 1068 in wild type cells) was set as 1. Results are presented as the means ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Phospho-proteomics, Imaging, Microscopy, Staining, Western Blot, Recombinant, Control, Software, Quantitative Proteomics, Mutagenesis

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Evaluation of EGFR downstream signaling in wild type cells and clones upon treatment with EGF. ( A ) Western blots were performed on whole cell lysates isolated from cells serum starved for 1 h and then incubated with recombinant EGF for 30 min in serum free media with 1% BSA. Control cells were incubated in serum free media for 30 min. Experiments were performed independently from at least 3 different passages of cells and representative blots are shown. Antibodies used in this work came from antibody panel ab283852 (Abcam, Cambridge, United Kingdom). ( B ) Quantification of Western blot results was performed using Li-cor quantification software. The results were normalized to protein expression in wild type cells not treated with EGF set at 100%. Data are presented as the mean ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Clone Assay, Western Blot, Isolation, Incubation, Recombinant, Control, Software, Expressing

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Evaluation of cell cycle distribution and expression of cell cycle regulator proteins. ( A ) Representative cell cycle profiles of wild type ME180 cells and mutant clones based on DAPI staining and incorporation of EdU. The usual horseshoe pattern cell distribution allowed separation of cell populations into G1, S and G2/M stage of the cell cycle. ( B ) Histogram showing cell cycle distribution in wild type cells and mutant clones based on flow cytometry data done on 5 independent experiments using cells from 5 different cell passages. The data are shown as means ± SD from five independent experiments including cells from three different cell cycle passages. ( C ) Western blots performed on whole cell lysates from wild type cells and mutant clones growing in cell culture or additionally treated with recombinant EGF for 30 min. Experiments were performed independently using at least three different cell passages and representative blots are shown. ( D ) Quantification of Western blot results was performed using Li-Cor quantification software and the results are presented values relative to protein expression in untreated wild type cells set as 100%. Data are presented as means ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Expressing, Mutagenesis, Clone Assay, Staining, Flow Cytometry, Western Blot, Cell Culture, Recombinant, Software

Journal: Nature Immunology

Article Title: E-selectin-mediated rapid NLRP3 inflammasome activation regulates S100A8/S100A9 release from neutrophils via transient gasdermin D pore formation

doi: 10.1038/s41590-023-01656-1

Figure Lengend Snippet: a , b , Caspase 1 cleavage was assessed in isolated human neutrophils resuspended in normal HBSS and HBSS with high [K + ] ex ( a ) and pretreated with K V 1.3 inhibitor PAP-1 ( b ; 50 nM) or vehicle control and stimulated with E-selectin for 10 min. The amount of processed caspase 1 (casp-1 p20 and casp-1 p22) was determined in the supernatants, and the amounts of procaspase 1 and GAPDH were determined in cell lysates ( n = 3 and 5 independent experiments, respectively). c , d , Bone marrow neutrophils from WT mice pretreated with PAP-1 (50 nM) or vehicle control ( c ) or from WT and Kcna3 −/− mice ( d ) were incubated with E-selectin or PBS (control) for 10 min ( n = 4 (control), 5 (PAP-1), 4 (WT) and 6 ( Kcna3 −/− ) mice per group); w/o, without. e , f , Bone marrow neutrophils from WT mice were incubated with PBS, E-selectin or ATP ( e ) or with PBS, E-selectin, nigericin or a combination of LPS and nigericin ( f ) for 10 min ( n = 5 (control ( e ), E-selectin ( e ), ATP ( e ) and nigericin ( f )), 9 (control ( f ), LPS and nigericin ( f )) and 8 (E-selectin ( f )) mice per group). g , Isolated human neutrophils were stimulated with PBS, E-selectin, MAdCAM-1 or endoglycan for 10 min ( n = 3 independent experiments). In c – g , supernatants were collected, and S100A8/S100A9 levels were analyzed by ELISA. h , i , IL-1β levels were analyzed by ELISA in the supernatants from WT bone marrow neutrophils stimulated with PBS, E-selectin, nigericin or a combination of LPS and nigericin for 10 min ( h ; n = 6 mice per group) or primed with PBS or LPS for 2.5 h and subsequently stimulated for 30 min with PBS, E-selectin or nigericin ( i ; n = 5 (control, LPS/nigericin) and 7 (LPS, LPS/E-selectin) mice per group). Data are presented as representative western blots and mean ± s.e.m. ( a and b ; data were analyzed by one-way ANOVA with a Tukey’s multiple comparison test or two-tailed paired Student’s t -tests, respectively) or mean ± s.e.m. ( c – i ; data were analyzed by two-way RM ANOVA with a Sidak’s multiple comparison test ( c and d ) or one-way ANOVA with Tukey’s multiple comparison tests ( e – i )).

Article Snippet: For human in vitro release assays, 5 × 10 5 neutrophils were stimulated with recombinant human E-selectin, recombinant human endoglycan, recombinant human MAdCAM (all 1 μg ml −1 ; R&D Systems) or PBS.

Techniques: Isolation, Control, Incubation, Enzyme-linked Immunosorbent Assay, Western Blot, Comparison, Two Tailed Test