Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: Elevated soluble CD14 (sCD14) concentration in the serum of cirrhotic patients. Concentration of sCD14 was measured in healthy controls (n=31), patients with chronic viral hepatitis (n=26) and patients with cirrhosis (n=50) as described in patients and methods. Data is presented graphically as Whisker box-plots

Article Snippet: For sCD14, human CD14 ELISA Development kit (R&D Systems, Abingdon, UK) was used following manufacturer’s instructions.

Techniques: Concentration Assay, Whisker Assay

Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: High correlation between the levels of human beta defensin-1 (hBD-1) and soluble CD14 (sCD14) in the hepatic veins of cirrhotic patients . Concentrations of hBD-1 and sCD14 were measured as described in patients and methods. Each dot corresponds to individual patients with cirrhosis (n=45). Analysis was performed in samples collected from peripheral veins (n=25, ) and from hepatic veins (n=20, )

Article Snippet: For sCD14, human CD14 ELISA Development kit (R&D Systems, Abingdon, UK) was used following manufacturer’s instructions.

Techniques:

Journal: Annals of Gastroenterology : Quarterly Publication of the Hellenic Society of Gastroenterology

Article Title: Systemic levels of human β-defensin 1 are elevated in patients with cirrhosis

doi:

Figure Lengend Snippet: Soluble CD14 (sCD14) and lipopolysaccharide binding protein (LBP) strongly correlate in serum of patients with cirrhosis. Concentrations of sCD14 were measured as described in Patients and Methods. Concentrations of LBP were measured by a commercially available ELISA, according to manufacturer’s instructions. Each dot corresponds to individual patients with cirrhosis (n=36). Analysis was performed in samples collected from peripheral veins

Article Snippet: For sCD14, human CD14 ELISA Development kit (R&D Systems, Abingdon, UK) was used following manufacturer’s instructions.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 infection is associated with intestinal permeability, systemic inflammation, and microbial dysbiosis in hospitalized COVID-19 patients

doi: 10.1101/2023.12.07.570670

Figure Lengend Snippet: Plasma concentrations for I-FABP ( A ), Zonulin ( B ), LBP ( C ), and sCD14 ( D ) measured by ELISA and circulating fatty acids propionic acid ( E ), decanoic acid ( F ), butyric acid ( G ), nonanoic acid ( H ), and isovaleric acid ( I ) measured by LC-MS/MS. Comparisons between groups were performed by Kruskal-Wallis tests, followed by Dunn post-hoc tests if adjusted p values were below 0.05. Pairwise comparisons between each variable were corrected separately for false discovery rate by the Benjamini-Hochberg method and adjusted p values <0.05 were considered significant.

Article Snippet: Gut barrier damage biomarkers, including LBP (cell sciences CKH113), sCD14 (R&D Systems QK383), zonulin (MyBioSource MBS706368), and I-FABP (R&D Systems DFBP20) were measured using commercially available assays according to manufacturer’s guidelines.

Techniques: Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: Monocyte subpopulations` content in healthy individuals and CRC patients.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques:

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: The distribution of CD163+ and CCR2+ peripheral blood monocytes in patients with colon and rectal cancers. Individual profiles of CCR2+ and CD163+ monocyte subsets for each patient with rectal and colon cancers. (A) , The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after NAC and after surgical resection in rectal cancer patients. (B) , The distribution of monocytes of classical (CD14+CD16-), intermediate (CD14+CD16+) and non-classical (CD14-CD16+) populations expressing CCR2 (upper panel) and CD163 (lower panel) is demonstrated before and after surgical resection in colon cancer patients. (C) , Associations of CCR2-expressing monocyte subsets with hematogenous and lymphatic metastasis in rectal cancer patients. (D) , Associations of CD163-expressing monocyte subsets with hematogenous and lymphatic metastasis in colon cancer patients. M 0 , metastasis-negative status, M 1 , metastasis-positive status. N 0 , lymph node-negative status, N 1-3 , lymph node-positive status.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse

doi: 10.3389/fimmu.2022.1080501

Figure Lengend Snippet: An activator of glycolysis PFKFB3 is overexpressed in colon cancer and is indicative for higher risk of tumor relapse in colon cancer but not rectal cancer. (A) , Сolon cancer tissue is massively infiltrated by PFKFB3-positive monocytes. IF/confocal microscopy analysis was performed for 10 colon tumor tissues. The infiltration of CD14+CD68+PFKFB3+ cells was found in all samples. Representative image is given from one patient. Scale bar corresponds to 50 µm in main image and 20 µm in zoom image. (B) , Spearman correlation coefficients between PFKFB3 expression, M2 macrophage gene expressions and predicted cell abundance scores, FDR<0.05. (C) , Predicted cell composition of CD45+ AOIs and hierarchical clustering of AOIs. (D) , Difference in monocyte and macrophage cell abundance scores between the CD45+ AOIs in colon and rectal cancers (the Mann-Whitney U test was applied). (E) , PFKFB3 gene expression is elevated in patients with recurrence and larger tumor size in colon cancer. Variance in PFKFB3 expression was stabilized via the variance stabilizing transformation (VST). (F) , PFKFB3 had prognostic significance for the DFS and OS. High-risk group had worse survival rates compared to low-risk group. ROC analysis and Kaplan–Meier method were applied.

Article Snippet: For immunofluorescence (IF) staining, tumor FFPE clinical samples were treated with xylol solution and blocked with 3% BSA in PBS for 45 min, incubated with a combination of primary antibodies for 1,5 h; washed, and incubated with a combination of appropriate secondary antibodies for 45 min. Anti-PFKFB3 rabbit monоclonal antibody (1:50, #ab181861, Abcam, USA); anti-CD68 monoclonal mouse antibody (1:100, #NBP2-44539, clone KP1, Novus Biologicals); anti-CD14 polyclonal sheep antibody (1:50, #BAF383, R&D Systems) were used.

Techniques: Confocal Microscopy, Expressing, MANN-WHITNEY, Gene Expression, Transformation Assay

Journal: Frontiers in Pharmacology

Article Title: Nano-in-Micro-Particles Consisting of PLGA Nanoparticles Embedded in Chitosan Microparticles via Spray-Drying Enhances Their Uptake in the Olfactory Mucosa

doi: 10.3389/fphar.2021.732954

Figure Lengend Snippet: Permeation of PLGA nanoparticles (520 nm; red) (A–E) and PLGA-chitosan nano-in-micro particles (NiMPs) (F–J) . Maximum intensity projection (MIP) of Confocal Laser Scanning Microscopy (CLSM) images stitched together from a z-stack, taken 15 min after application to the apical side of porcine olfactory mucosa. Cell nuclei stained with DAPI (blue). Red arrows mark nanoparticles (red) within the tissue. CD14 (green) labelled with anti-mouse Alexa Fluor ® 647 secondary antibody (A, B, F, G) . NF-H (cyan) labelled with anti-chicken FITC secondary antibody (D, E) . Chitosan labeled with fluorescein (cyan) (H) . Overlapping signals of red and green appear yellow (F) due to co-localization of PLGA nanoparticles (red) and chitosan (green). PLGA nanoparticles (single red channel) displayed in . LP, Lamina propria; BL, Basal cell layer; PL, Particle layer; EL, Epithelial cell layer.

Article Snippet: To visualize the initial immune reaction, CD14 + cells were stained via CD14 primary antibody (#NB100-77758, Novus Biologicals, Littleton, United States) and goat anti-mouse secondary IgG (H+L) Alexa Fluor ® 647 (#A-21235, Thermo Fisher Scientific, Waltham, United States).

Techniques: Confocal Laser Scanning Microscopy, Staining, Labeling

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Maternal and Foetal Cellular Immune Responses in Dams Infected With High- and Low- Virulence Isolates of Neospora caninum at Mid-Gestation

doi: 10.3389/fcimb.2021.684670

Figure Lengend Snippet: List of antibodies used for analysis of cell subsets in PBMC, ileofemoral lymph node, fetal thymus and spleen.

Article Snippet: CD14 , FITC , Bio-Rad Laboratories (Pleasanton, CA, USA) , Mouse anti-bovine , Monoclonal , CC-G33 , MCA2678F.

Techniques:

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Maternal and Foetal Cellular Immune Responses in Dams Infected With High- and Low- Virulence Isolates of Neospora caninum at Mid-Gestation

doi: 10.3389/fcimb.2021.684670

Figure Lengend Snippet: Relative percentage of immune cells in a PBMC population from Nc-Spain7- and Nc-Spain1H-infected heifers. Graphs indicate the relative percentage of cells positive for CD21, WC1, CD4, CD8, CD14 and CD335 surface markers, and CD4+/CD8+ ratio in PBMC obtained from uninfected heifers (G-Control) and heifers challenged with Nc-Spain7 (G-NcSpain7) and Nc-Spain1H (G-NcSpain1H) tachyzoites. The results are represented as the mean + SD. Asterisks indicate significant differences in G-NcSpain1H versus G-Control comparison (blue), G-NcSpain7 versus G-Control (red) and G-NcSpain1H versus G-NcSpain7 (black). **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05.

Article Snippet: CD14 , FITC , Bio-Rad Laboratories (Pleasanton, CA, USA) , Mouse anti-bovine , Monoclonal , CC-G33 , MCA2678F.

Techniques: Infection, Control, Comparison

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Maternal and Foetal Cellular Immune Responses in Dams Infected With High- and Low- Virulence Isolates of Neospora caninum at Mid-Gestation

doi: 10.3389/fcimb.2021.684670

Figure Lengend Snippet: Relative percentage of immune cells in iliofemoral lymph nodes from Nc-Spain7- and Nc-Spain1H-infected heifers. Graphs indicate the relative percentage of cells positive for CD21, WC1, CD4, CD8, CD14 and CD335 surface markers, and CD4+/CD8+ ratio surface markers in the population of immune cells obtained from the iliofemoral lymph node of uninfected heifers (G-Control) and heifers challenged with Nc-Spain7 (G-NcSpain7) and Nc-Spain1H (G-NcSpain1H) tachyzoites culled at 10 or 20 dpi. The results are represented as the mean + SD. Asterisks indicate significant differences. ** P < 0.01, * P < 0.05.

Article Snippet: CD14 , FITC , Bio-Rad Laboratories (Pleasanton, CA, USA) , Mouse anti-bovine , Monoclonal , CC-G33 , MCA2678F.

Techniques: Infection, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Maternal and Foetal Cellular Immune Responses in Dams Infected With High- and Low- Virulence Isolates of Neospora caninum at Mid-Gestation

doi: 10.3389/fcimb.2021.684670

Figure Lengend Snippet: Immune cell populations in the spleen of foetuses from Nc-Spain7 and Nc-Spain1H infected heifers at 10 and 20 dpi. Graphs indicate the relative percentage of cells positive for CD21, WC1, CD4, CD8, CD14 and CD335 surface markers, double positive (DP) CD4+CD8+ cells and CD4+/CD8+ ratio, in the population of immune cells obtained from the spleen of foetuses from uninfected heifers (G-Control) and heifers challenged with Nc-Spain7 (G-NcSpain7) and Nc-Spain1H (G-NcSpain1H) tachyzoites culled at 10 or 20 dpi. The results are represented as the mean + SD. Asterisks indicate significant differences. ** P < 0.01, * P < 0.05.

Article Snippet: CD14 , FITC , Bio-Rad Laboratories (Pleasanton, CA, USA) , Mouse anti-bovine , Monoclonal , CC-G33 , MCA2678F.

Techniques: Infection, Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Maternal and Foetal Cellular Immune Responses in Dams Infected With High- and Low- Virulence Isolates of Neospora caninum at Mid-Gestation

doi: 10.3389/fcimb.2021.684670

Figure Lengend Snippet: Immune cell populations in the thymus of foetuses from Nc-Spain7 and Nc-Spain1H infected heifers at 10 and 20 dpi. Graphs indicate the relative percentage of cells positive for CD21, WC1, CD4, CD8, CD14 and CD335 surface markers, double positive (DP) CD4+CD8+ cells and CD4+/CD8+ ratio, in the population of immune cells obtained from the thymus of foetuses from uninfected heifers (G-Control) and heifers challenged with Nc-Spain7 (G-NcSpain7) and Nc-Spain1H (G-NcSpain1H) tachyzoites culled at 10 or 20 dpi. The results are represented as the mean + SD. Asterisks indicate significant differences. * P < 0.05.

Article Snippet: CD14 , FITC , Bio-Rad Laboratories (Pleasanton, CA, USA) , Mouse anti-bovine , Monoclonal , CC-G33 , MCA2678F.

Techniques: Infection, Control

Journal: iScience

Article Title: Dietary fiber reduces mortality from secondary sepsis in a murine model of Clostridioides difficile infection

doi: 10.1016/j.isci.2026.115258

Figure Lengend Snippet: Plasma markers of systemic inflammation and kidney injury in CDI mice across diets (A) PCoA of Canberra distances of mean-normalized sepsis/immune marker concentrations. Points are colored by diet, and shapes indicate whether mice were humanely euthanized due to clinical sickness or survived until the experimental endpoint. Vectors show the correlation of each measured factor with PC1 and PC2, with the vector length indicating the relative strength of the correlation. Only statistically significant vectors are shown (multiple regression with Benjamini-Hochberg multiple test correction, p.adj. < 0.05). (B) Plasma concentration of each marker that significantly differed between diets (blood urea nitrogen (BUN), and immune factors sCD14, CXCL1, IL-10, IL-1B, IL-6, and TNF-a) at sacrifice. Pairwise comparisons of concentrations between diets were calculated using Kruskal-Wallis and Dunn’s post hoc tests, with p -value corrections conducted via Benjamini and Hochberg. Boxplot lines (from top to bottom) depict the 75 th , 50 th (median), and 25 th percentiles, with lines extending from the top/bottom of the boxplot indicating the largest/smallest observation within ±1.5∗IQR (inter-quartile range). P-value significance (∗∗∗∗: p < 0.0001, ∗∗∗: p < 0.001, ∗∗: p < 0.01, and ∗: p < 0.05).

Article Snippet: Soluble CD14 (sCD14) levels were quantified using the Quantikine Mouse sCD14 ELISA Kit (Catalog No. MC140, R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Clinical Proteomics, Marker, Plasmid Preparation, Concentration Assay

Journal:

Article Title: LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 Expression in Human Lymphatic Endothelium

doi: 10.1369/jhc.7A7299.2007

Figure Lengend Snippet: Expression of toll-like receptor 4 (TLR4), MD-2, and CD14. (A) Immunohistochemical analysis. The cell membrane was visualized with rhodamine-conjugated Con A (Con A, in red), and the reaction products to antibodies for TLR4, MD-2, and CD14 were visualized with an AF488-conjugated second antibody (AF488, in green). Merged images indicate that the expression of TLR4, MD-2, and CD14 was immunohistochemically observed in the cytoplasm and on the cell membrane of LEC, and that the three were detected on the membrane of HEK-293 cells stably transfected with the human TLR4, MD-2, and CD14 genes (tHEK), and not in the negative control HEK-293 cells (HEK). Bar = 100 μm. (B) RT-PCR analysis. RT-PCR products for TLR4, MD-2, and CD14 mRNAs were not detected in HEK-293 but were detected in LEC and in tHEK. MW, molecular weight marker.

Article Snippet: The cells were immunostained with 1 μg/ml of rabbit antiserum to human Prox1 (AngioBio Co.; Del Mar, CA) and of monoclonal antibodies to human podoplanin (AngioBio), VCAM-1, and ICAM-1 (R and D Systems, Inc.; Minneapolis, MN), and also immunostained with 5 μg/ml of monoclonal antibodies to TLR4, MD-2, and CD14 (R and D Systems).

Techniques: Expressing, Immunohistochemical staining, Membrane, Stable Transfection, Transfection, Negative Control, Reverse Transcription Polymerase Chain Reaction, Molecular Weight, Marker

Journal:

Article Title: LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 Expression in Human Lymphatic Endothelium

doi: 10.1369/jhc.7A7299.2007

Figure Lengend Snippet: Expression of genes for lymphatic endothelial markers, and TLR-associated and leukocyte adhesion molecules in human neonatal dermal lymphatic microvascular endothelial cells with LPS treatments

Article Snippet: The cells were immunostained with 1 μg/ml of rabbit antiserum to human Prox1 (AngioBio Co.; Del Mar, CA) and of monoclonal antibodies to human podoplanin (AngioBio), VCAM-1, and ICAM-1 (R and D Systems, Inc.; Minneapolis, MN), and also immunostained with 5 μg/ml of monoclonal antibodies to TLR4, MD-2, and CD14 (R and D Systems).

Techniques: Expressing, Control, Marker, Activation Assay

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Generation of A431D cells that stably express CD148 forms (WT, Q276P/R326Q). (A) DNA sequence of mutated CD148 cDNA, confirming Q276P and R326Q mutations. (B) CD148 surface expression were examined in A431D‐CD148 (WT, Q276P/R326Q) cells and the control A431D cells that was generated by the infection of empty retrovirus (Mock) by flow cytometry using a PE‐conjugated anti‐CD148 antibody. Representative results of four independent experiments are shown. Mean fluorescence intensity is also shown. The fluorescence intensity of CD148 WT cells is expressed as 1.0. A431D‐CD148 WT and Q276P/R326Q cells express comparable levels of CD148. A431D‐Mock cells show no CD148 expression. (C) Expression of CD148 in A431D‐CD148 (WT, Q276P/R326Q) cells was also examined by Western blotting using anti‐CD148 or anti‐HA antibodies. Fifty micrograms of protein cell lysates were loaded into each well. The loading of proteins was assessed by anti‐β actin Western blot. The ratios of CD148 or HA to β actin are also shown. The ratios in A431D‐CD148 WT cells are expressed as 1.0. Representative results of four independent experiments are shown. (D) Immunolocalization of CD148 or HA epitope (both red) were examined in A431D‐Mock and A431D‐CD148 (WT, Q276P/R326Q) cells. The antibody against CD148 extracellular domain was used. Representative results of four independent experiments are shown. Scale bar = 25 μm. CD148 WT and Q276P/R326Q are similarly localized to the cell membrane (arrowheads). In anti‐HA immunostaining, there is perinuclear CD148 presence that is generally observed in stable cells

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Stable Transfection, Sequencing, Expressing, Control, Generated, Infection, Flow Cytometry, Fluorescence, Western Blot, Membrane, Immunostaining

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Cell proliferation rate in A431D‐CD148 (WT, Q276P/R326Q) cells. Cells (1.0 × 10 3 ) were plated in 96‐well plates and serum starved (0.1% FBS) for overnight (day 1). Cells were then cultured in growth medium supplemented with 2.5% FBS. Cell number was assessed at day 1, 2, 3 and 4. Data are means ± SEM of quadruplicate determinations. Representative data of four independent experiments is shown. ** p < .01. A431D‐CD148 (WT, Q276P/R326Q) cells showed lower cell proliferation rates than A431D‐Mock cells. No significant difference was observed between A431D‐CD148 WT and Q276P/R326Q cells

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Cell Culture

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Expression of epidermal growth factor receptor (EGFR) and EGF‐induced ERK phosphorylation in A431D‐CD148 (WT, Q276P/R326Q) cells. (A) Cell surface expression of EGFR in A431D‐CD148 (WT and Q276P/R326Q) and A431D‐Mock (control) cells were examined by flow cytometry using a PE‐conjugated anti‐human EGFR antibody. HEK293 cells that do not express EGFR were used as a negative control. Mean fluorescence intensity is also shown (bottom right). The fluorescence intensity of A431D‐Mock cells is expressed as 1.0. Representative results of three independent experiments are shown. All three A431D cells showed comparable level of EGFR expression. (B) EGF‐induced ERK1/2 phosphorylation was examined in A431D‐CD148 (WT and Q276P/R326Q) and A431D‐Mock cells by ELISA as described in Section . Cells were treated with 10 ng/ml EGF for 5, 10, 15, and 30 min. The cells cultured in serum starved (SS) condition (0.1% FBS medium) for 30 min were used as a control. The p‐ERK/ERK ratios were normalized to serum‐starved (30 min) A431D‐Mock cells. Representative results of five independent experiments are shown. Data are means ± SEM of quadruplicate measurements. *** p < .001

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Expressing, Phospho-proteomics, Control, Flow Cytometry, Negative Control, Fluorescence, Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Immunoblotting to assess EGF‐induced epidermal growth factor receptor (EGFR) and ERK1/2 phosphorylation in A431D‐CD148 (WT, Q276P/R326Q) cells. (A) and (B) EGF‐induced EGFR and ERK1/2 phosphorylation was examined in A431D‐CD148 (WT, Q276P/R326Q) and A431D‐Mock cells by Western blotting as described in Section . Cells were plated in 100 mm dishes, serum was reduced (0.5% FBS), then cells were treated with 10 ng/ml EGF for 5, 10, 15, 30 min. The cells cultured in serum starved (SS) condition (0.1% FBS medium) for 30 min were used as a negative control. The p‐EGFR/EGFR ratios were normalized to A431D‐Mock cells treated with EGF for 5 min as EGFR phosphorylation was undetectable in serum starved (0.1% FBS) cells. The p‐ERK/ERK ratios were normalized to serum‐starved A431D‐Mock cells. Representative results of five independent experiments are shown

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: Western Blot, Phospho-proteomics, Cell Culture, Negative Control

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: EGF‐induced cell proliferation in A431D‐CD148 (WT, Q276P/R326Q) cells. Cells (1.2 × 10 3 ) were plated in 96‐well plates (day 0), then serum starved (0.1% FBS) for overnight (day 1). Cells were then treated with 60 ng/ml, 120 ng/ml, and 240 ng/ml EGF in growth medium supplemented with 0.3% FBS. Cell number was assessed at day 1 (before EGF stimulation) and day 3. Representative results of four independent experiments are shown. Data are means ± SEM of quadruplicate determinations. ** p < .01, * p < .05

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques:

Journal: Cancer Reports

Article Title: The effects of CD148 Q276P / R326Q polymorphisms in A431D epidermoid cancer cell proliferation and epidermal growth factor receptor signaling

doi: 10.1002/cnr2.1566

Figure Lengend Snippet: Effects of TSP1 on cell proliferation in A431D‐CD148 (WT, Q276P/R326Q) cells. Cells (1.0 × 10 3 ) were plated in 96‐well plates (day 0), then serum starved (0.1% FBS) for overnight (day 1). Cells were then treated with 1, 10, 20 nM of TSP1 in growth medium supplemented with 2.5% FBS. Cell number was assessed at day 1 (before TSP1 stimulation) and day 4. Representative results of four independent experiments are shown. Data are means ± SEM of quadruplicate determinations. ** p < .01

Article Snippet: The cells were dissociated with 0.05%Trypsin, 0.53 mM EDTA (Corning, Manassas, VA), washed with PBS containing 0.5% Bovine Serum Albumin (BSA) (Sigma Aldrich), resuspended in cold 0.5% BSA‐PBS, and 2 × 10 5 cells were incubated with 5 μl of PE‐conjugated anti‐human CD148 mouse monoclonal (clone 143‐41, R&D Systems) or 10 μl of PE‐conjugated anti‐human EGFR (BD Biosciences) for 45 min at 4°C.

Techniques: