Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Immunostaining of CARD8 in non-atherosclerotic arteries. CARD8 expression was found in the endothelial layer of intima and smooth muscle cells in the media of non-atherosclerotic artery (Left: Artery from colon tissue, Magnification, × 30; Right: Popliteal artery, Magnification, × 10). Scale bar: 100 μm. Full images are available as Supplementary material.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Immunostaining, Expressing

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Representative image of immunostaining of human carotid atherosclerotic plaque. Expression of CARD8 staining (brown/DAB) was evident in endothelial cells is shown using the arrows ( A ), moderately in smooth muscle cells (red/SMA) (as shown with the arrow ( B ) and CD68 (red) positive macrophages (as shown with the arrow ( C ). Expression of CARD8 staining (brown/DAB) in the SMA ( D ) and CD68 ( E ) positive cells in the intimal region of lesions. Left Panel: Magnification, × 2 and Scale bar: 500 μm; Right Panel: Magnification, × 30 (D&E × 20) and Scale bar: 50 μm. Full images are available as Supplementary material.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Immunostaining, Expressing, Staining

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Correlation analysis using microarray data from BiKE study. CARD8 expression positively correlated with the expression of CD163 ( A ), and VWF ( B ), in human carotid atherosclerotic plaques. Solid line represent the trend line and the dashed lines represent the 95% confidence intervals for the trend lines.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Microarray, Expressing

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Expression of CARD8 mRNA and protein in control and CARD8 knock down HUVECs. The knock down of CARD8 significantly reduced the mRNA ( A ) and protein levels ( B ) of CARD8. Representative protein bands are shown in ( C ). The full image of ( C ) is shown as Supplementary data. Data is presented as mean ± SD for n = 3–6 in each group. p value **** p < 0.0001 is compared to control.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Expressing, Control, Knockdown

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Subcellular localization of CARD8 in HUVEC. CARD8 expression in HUVECs treated with control/scramble siRNA ( A ) or CARD8 siRNA ( B ). CARD8 expression is indicated in green , f-actin in red and nucleus are stained blue. Magnification, × 40 and Scale bar: 20 μm. Full images are available as Supplementary material.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Expressing, Control, Staining

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Differentially regulated proteins after CARD8 knock down (CARD8 KD) in HUVECs using Olink proteomics panels. Volcano plot displaying differential protein expression in the lysate (CVD II ( A ) and CVDIII ( B ) panels) and in the culture medium [Inflammation panel ( C )]. Colors represent FDR levels (red, FDR ≤ 1%; green, FDR ≤ 5%; blue, FDR ≤ 10%; black, FDR > 10%). The labeled dots represent proteins that were differentially expressed in CARD8 knock down versus control HUVECs (FDR ≤ 10%). ( D ) The protein–protein interaction network as analyzed by String software. Proteins in the STRING software corresponds to the following: TNFRSF10B = TRAIL-R2; F2R = PAR-1; THBD = TM; TNFRSF1A = TNF-R1; TNFSF12 = TWEAK; HSPB1 = HSP27; CCL2 = MCP-1; PLAT = tPA; CXCL8 = IL-8; EIF4EBP1 = 4E-BP1; CCL7 = MCP3; ANGPT1 = ANG1: LDLR = LDL receptor; SIRPA = SHPS1; TNFRSF11B = OPG; PLAU = uPA. The red, violet and green nodes represents proteins involved in inflammatory response, cytokine-mediated signaling pathway and immune system process respectively. The colored lines represent the different possible association between the proteins. A red line indicates the presence of fusion evidence; a green line indicates neighborhood evidence; a blue line indicates co-occurrence evidence; a purple line indicates experimental evidence; a yellow line indicates text-mining evidence; a light blue line indicates database evidence; and a black line indicates co-expression evidence.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Knockdown, Expressing, Labeling, Control, Software

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Gene expression levels of selected genes regulated by CARD8. The knock down of CARD8 significantly reduced the expression of CXCL1 ( p = 0.0034), IL6 ( p < 0.0001), CXCL6 ( p < 0.0001), and MCP-1 ( p = 0.0066), when compared to the control. The expression of PDGF-A ( p = 0.0066) was significantly upregulated after the knock down of CARD8 when compared to control. Data are representative of samples from 3 independent experiments and displayed as mean ± SD.

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Expressing, Knockdown, Control

Journal: Scientific Reports

Article Title: Expression of CARD8 in human atherosclerosis and its regulation of inflammatory proteins in human endothelial cells

doi: 10.1038/s41598-020-73600-4

Figure Lengend Snippet: Correlation between gene expression of CARD8 and genes associated to inflammatory response and cell migration in human atherosclerotic lesions. CARD8 correlated with ( A ) CXCL1 (r = 0.48, p = 0.000000088); ( B ) CXCL6 (r = 0.31, p = 0.0022); ( C ) MCP-1 (r = 0.28, p = 0.011); ( D ) ALCAM (r = 0.26, p = 0.026); and ( E ) PDGFA (r = −0.38, p = 0.000062); ( F ) IL6 (r = 0.29; p = 0.00082).

Article Snippet: The staining was performed using primary antibodies against CARD8 (PAB0281; Abnova Corp, Taipei City, Taiwan; 1:1000/Nordic BioSite AB, Täby, Sweden), CD68 (NCL‐L‐CD68, Novacastra, Newcastle, UK; 1:50), VWF (M0616; Dako, Glostrup, Denmark; 1:50) and smooth muscle actin (SMA; M0851; Dako; 1:500) diluted in Da Vinci Green (Biocare Medical) and incubated for 1 h at room temperature.

Techniques: Expressing, Migration

Journal: Archives of Industrial Hygiene and Toxicology

Article Title: Associations of NLRP3 and CARD8 Gene Polymorphisms with Alcohol Dependence and Commonly Related Psychiatric Disorders: a Preliminary Study

doi: 10.2478/aiht-2021-72-3432

Figure Lengend Snippet: NLPR3 rs35829419 and CARD8 rs2043211 genotype distribution by groups

Article Snippet: The three groups significantly differed in the distribution of the CARD8 rs2043211 genotypes (P=0.049; chi-squared=9.557; df=4) but not in the distribution of the NLPR3 rs35829419 genotypes.

Techniques:

Journal: Archives of Industrial Hygiene and Toxicology

Article Title: Associations of NLRP3 and CARD8 Gene Polymorphisms with Alcohol Dependence and Commonly Related Psychiatric Disorders: a Preliminary Study

doi: 10.2478/aiht-2021-72-3432

Figure Lengend Snippet: Associations between CARD8 rs2043211 genotypes and alcohol-related mental disorders

Article Snippet: The three groups significantly differed in the distribution of the CARD8 rs2043211 genotypes (P=0.049; chi-squared=9.557; df=4) but not in the distribution of the NLPR3 rs35829419 genotypes.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: Caspase Recruitment Domain-containing Protein 8 (CARD8) Negatively Regulates NOD2-mediated Signaling

doi: 10.1074/jbc.m110.127480

Figure Lengend Snippet: FIGURE 1. CARD8 co-localizes and physically interacts with NOD2. A, HeLa cells expressing CFP-CARD8 and YFP-NOD2 were subjected to fluorescence microscopy. Regions of co-localization are indicated by white arrows. B, HEK cells were co-transfected with expression constructs encoding for FLAG-NOD2 and CARD8. 24 h after transfection NOD2 complexes were immunoprecipitated with anti-FLAG or anti-CARD8 antibody. Co-precipitating CARD8 (or FLAG-NOD2) was detected by immunoblotting with the respective antibodies. C and D, HEK cells were co-transfected with GFP-CARD8 and the indicated FLAG-NOD2 expression constructs encoding for full-length NOD2, LRR, NBD, or the CARDs. Co-precipitating FLAG-NOD2 was detected with anti-FLAG monoclonal anti- body. Expression of GFP-CARD8 and FLAG-NOD2 constructs is shown in the lower insets.

Article Snippet: The antibodies used for immunoprecipitation andWestern blotting were monoclonal mouse anti-FLAG antibody (M2, Sigma-Aldrich), anti-Myc antibody (Clontech, Palo Alto, CA), anti-GFP antibody (Clontech, Palo Alto, CA); anti-CARD8 antibody (ProSci, Poway, CA), and -actin (Sigma Aldrich).

Techniques: Expressing, Fluorescence, Microscopy, Transfection, Construct, Immunoprecipitation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Caspase Recruitment Domain-containing Protein 8 (CARD8) Negatively Regulates NOD2-mediated Signaling

doi: 10.1074/jbc.m110.127480

Figure Lengend Snippet: FIGURE 2. CARD8 is up-regulated in intestinal inflammation and co-localizes with NOD2 in the mucosa of Crohn disease patients. A, protein extracts were prepared from biopsies from the colonic mucosa of healthy individuals (HN) and patients with Crohn Disease (CD infl.). B, cryosections of mucosal biopsies were stained using appropriate antibodies to detect endogenous NOD2 (green channel, FITC) and CARD8 (red channel, Cy3), respectively. In addition, nuclei were stained using DAPI (blue channel). The merged pictures show overlapping expression patterns of endogenous NOD2 and CARD8 (indicated by arrows).

Article Snippet: The antibodies used for immunoprecipitation andWestern blotting were monoclonal mouse anti-FLAG antibody (M2, Sigma-Aldrich), anti-Myc antibody (Clontech, Palo Alto, CA), anti-GFP antibody (Clontech, Palo Alto, CA); anti-CARD8 antibody (ProSci, Poway, CA), and -actin (Sigma Aldrich).

Techniques: Staining, Expressing

Journal: Journal of Biological Chemistry

Article Title: Caspase Recruitment Domain-containing Protein 8 (CARD8) Negatively Regulates NOD2-mediated Signaling

doi: 10.1074/jbc.m110.127480

Figure Lengend Snippet: FIGURE3.CARD8increasesbacterialcytoinvasionandsuppressesNOD2-inducedNF-BactivityandsecretionofIL-1andIL-8.A,CARD8and/orNOD2 were expressed in HEK cells following infection with L. monocytogenes and gentamicin-mediated elimination of extracellular bacteria. Relative cytoinvasion was calculated as percentage of CFUs found in lysates of untransfected cells. Actual numbers of CFU are shown in parentheses. **, p 0.01; data are representative of three independent experiments (n 3). B, NOD2 and CARD8 were expressed as indicated in HEK cells and stimulated with MDP (24 h, 1 g/ml). Data are expressed as relative luciferase activity. C, knockdown of CARD8 gene expression by siRNA. Cells were transfected with a pool of specific siRNAs targeting CARD8 or irrelevant control siRNA with the indicated amounts. Efficiency of knock-down was analyzed using RT-PCR. D, cells were transfected with siRNA in combination with a NOD2 expression construct. After stimulation with MDP (20 g/ml) for 24 h relative luciferase activity was determined by normalization of luciferase activity to protein content. E–H, FLAG-NOD2, GFP-CARD8, and GFP-CARD8-(1–320) (FIIND-domain) were expressed in HEK cells as indicated and treated with MDP (24 h, 10 g/ml) before levels of cytokines were determined. All data represent the mean S.D. (n 3). *, p 0.05; **, p 0.01.

Article Snippet: The antibodies used for immunoprecipitation andWestern blotting were monoclonal mouse anti-FLAG antibody (M2, Sigma-Aldrich), anti-Myc antibody (Clontech, Palo Alto, CA), anti-GFP antibody (Clontech, Palo Alto, CA); anti-CARD8 antibody (ProSci, Poway, CA), and -actin (Sigma Aldrich).

Techniques: Infection, Bacteria, Luciferase, Activity Assay, Knockdown, Gene Expression, Transfection, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Construct

Journal: Journal of Biological Chemistry

Article Title: Caspase Recruitment Domain-containing Protein 8 (CARD8) Negatively Regulates NOD2-mediated Signaling

doi: 10.1074/jbc.m110.127480

Figure Lengend Snippet: FIGURE 4. CARD8 interferes with MDP-induced NOD2 oligomerization. FLAG- and Myc-tagged full-length NOD2 and the CARD8-(1–320) (FIIND domain of CARD8) were expressed in HEK cells and self-association of NOD2 was determined in absence or presence of MDP (10 g/ml) by immunopre- cipitation. Total lysates were immunoblotted with anti-FLAG, anti-GFP, and anti-Myc antibodies (lower three blots). Each gel is representative of at least three independent experiments.

Article Snippet: The antibodies used for immunoprecipitation andWestern blotting were monoclonal mouse anti-FLAG antibody (M2, Sigma-Aldrich), anti-Myc antibody (Clontech, Palo Alto, CA), anti-GFP antibody (Clontech, Palo Alto, CA); anti-CARD8 antibody (ProSci, Poway, CA), and -actin (Sigma Aldrich).

Techniques: