Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: A) A549 subpopulations collected from different cell growth stages (as indicated) were stained for CEACAM1 with mAb clone 283340, for CEACAM5 with Col-1, for CEACAM6 with mAb 9A6 and for CEACAM7 with BAC2 (thick line). The background fluorescence was determined by incubating the cells with control IgG antibody instead of primary anti-CEACAM antibody (thin line). Subsequently, samples were analyzed by flow cytometry revealing CEACAM1 expression in confluent A549-NT and A549-T cells. Additionally, a minor fraction of confluent A549-T cells expressed CEACAM6. The data shown are representative of three independent experiments. B) Determination of different CEACAMs in whole cell lysates of A549 cells cultured in the non-confluent log phase, the confluent phase and the spheroidal unanchored growing cells by immunoblotting with mAb specific for CEACAM1, CEACAM5 and CEACAM6. The detection of beta-actin served as a loading control. The data shown are representative of three different experiments. C) Soluble CEACAM1, CEACAM5 and CEACAM6 forms are released in the cell culture supernatant of confluent A549 cells. The released CEACAM1, CEACAM5 and CEACAM6 molecules were quantified by specific sandwich ELISA as described in the section. The mean values ± SD (*p≤0.005) were determined from triplicates. The experiment was repeated twice.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Staining, Fluorescence, Control, Flow Cytometry, Expressing, Cell Culture, Western Blot, Sandwich ELISA

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: A) For quantification of CEACAM1 cell surface expression in confluent and log phase cultured A549 cells, samples were analyzed by flow cytometry utilizing the QuiFiKit approach as described in . Briefly, cells were stained for CEACAM1 with mAb clone 283340. Fluorescence was quantitated using QuiFiKit calibration-beads as described in . The data shown are means ± SD (*p≤0.007) of three different experiments. B) Immunoblot analyses of confluent and proliferating A549 cell lysates were done as described in using monospecific mAbs directed against CEACAM1 (clone 283340), CEACAM5 (Col-1), and CEACAM6 (9A6), respectively and visualized by HRP-coupled secondary antibody and ECL detection. Beta-actin served as a loading control. The blots shown are representative of three separate experiments. C) The fraction of CEACAM1 expressing A549 cells increased over time as cells were kept in the confluent state. To analyze the CEACAM1 expression in A549 cells grown to confluence (day 0), plus 1 day, plus 3 days, plus 5 days and plus 7 days, cells were stained with mAb clone 283340 (thick line) and isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Subsequently, samples were measured by flow cytometry. The data shown are representative of three different experiments.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Expressing, Cell Culture, Flow Cytometry, Staining, Fluorescence, Western Blot, Control

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: RT of RNA isolated from confluent A549 cells were applied to four different PCR reactions using primer pairs specific four each of the four CEACAM1 isoforms. Products corresponding to CEACAM1-4L (266 bp), CEACAM1-4S (245 bp), CEACAM1-3L (177 bp), and CEACAM1-3S (145 bp), respectively, were amplified. The size of oligonucleotide markers is shown on the left.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Isolation, Amplification

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: Characterization of the CEACAM1 isoform pattern as determined by quantitative RT-PCR of the two A549 subpopulations.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: A) Analyses of the cell surface expression of different CEACAMs in the non-confluent log phase cultured parental A549 cells stably transfected with an empty vector (a), CEACAM1-4L (b), the mutant form of the intracellular ITIM motif CEACAM1-4L-Y459F/Y486F (c) and CEACAM5 (d). Samples were analyzed by flow cytometry using mAbs that specifically bind the different CEACAMs (thick line) or isotype matched control antibody (thin line) followed by FITC-conjugated secondary antibody. Data show one of three different, representative stably transfected A549 clones. B) Representative phase contrast images demonstrating the morphology of A549 cells stably transfected with empty vector (a), plasmids encoding for CEACAM1-4L (b), the ITIM mutant form CEACAM1-4L(Y459F/Y486F) (c), CEACAM5 (d) and CEACAM1-4S (e). Bar, 50 µm. C) Percentage of viable cells determined by the flow cytometry based annexin V-FITC/PI approach as described in using cell spheroidals and aggregates harvested from the culture supernatant of wild type (wt) A549 cells and A549 cells transfected with control vector, CEACAM1-4L (Y459F/Y486F) or A549-CEACAM5. Data are shown as the percentage of viable cells as the mean of three independent experiments +/− Standard deviation. D) Growth properties of control vector transfected and CEACAM1-4L transfected A549 cells as determined by the MTS based method as described in . A549- vector (white circles) and A549-CEACAM1 transfected cells (black circles) were seeded into in 96-well cell culture plates at a density of 25,000 cells/well. Following standard cell culture for 1, 2, 3, 4 and 5 days, respectively, the tetrazolium compound MTS was added and the samples were incubated at 37°C in a humidified, 5% CO2 atmosphere for 4 h. Experiments were performed in triplicate and results presented are expressed as means of OD 490 nm ± SD (*p≤0.005). The data show one representative result of three independent repeats of the experiment.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Expressing, Cell Culture, Stable Transfection, Transfection, Plasmid Preparation, Mutagenesis, Flow Cytometry, Control, Clone Assay, Standard Deviation, Incubation

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: A) The expression of the proliferation marker Ki67 is limited to A549-T cells that also expressed CEACAM6 on their cell surface. Confluent CEACAM6-negative and CEACAM6-positive A549-T cells were separated by mAb 9A6 loaded magnetic protein G microbeads and μMAS magnetic sorting columns (Miltenyi Biotec) as described in . Immunoblot analysis of confluent CEACAM6-negative and CEACAM6-positive A549-T cell lysates was performed applying a Ki67 specific antibody followed by HRP-coupled secondary antibody and ECL detection. Beta-actin served as a loading control. The data shown are representative of three separate experiments. B) Cell cycle analysis of CEACAM6 negative and CEACAM6 positive A549-T cells. CEACAM6-negative and CEACAM6-positive A549 cells were fixed in 80% ethanol and stained with propidium iodide, and analyzed by flow cytometry as described in “ .” The DNA content in the different cell fractions is given in arbitrary units on the X-axis. Cells in the G2-M phase (second peak) contained twice as much DNA as cells in the G0-G1 phase (first peak). Cells between the peaks represent cells in the S-phase. The relative proportions of cells in the various phases are shown above the DNA profiles. Filled curve, CEACAM6 negative A549 cells; thick curve, CEACAM6 positive A549 cells. C) Confluent control sh-plasmid transfected A549-T (A549-shControl) and shCEACAM6 transfected A549-T cells (A549-shCC6) were stained for CEACAM1 with mAb clone 283340 and for CEACAM6 with mAb 9A6 (thick lines). The background fluorescence was determined by incubating the cells with control IgG antibody (thin lines). Samples were analyzed by flow cytometry. Compared to A549-shControl cells, A549-shCC6 cells completely lacked CEACAM6 expression, but continued to express CEACAM1. D) Phase contrast images of (a) control sh-plasmid transfected A549-T and (b) shCEACAM6 transfected A549-T cells. Confluent control sh-plasmid transfected A549-T piled up and formed unanchored spheroidal cell aggregates on top of the monolayer revealing insufficient contact inhibition. (b) In contrast, A549-shCC6 cells formed well spread monolayers without detection of unanchored, spheroidal cell growth indicative tight contact inhibition. Bar, 50 µm.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques: Expressing, Marker, Western Blot, Control, Cell Cycle Assay, Staining, Flow Cytometry, Plasmid Preparation, Transfection, Fluorescence, Inhibition

Journal: PLoS ONE

Article Title: Deregulation of the CEACAM Expression Pattern Causes Undifferentiated Cell Growth in Human Lung Adenocarcinoma Cells

doi: 10.1371/journal.pone.0008747

Figure Lengend Snippet: Primer sequences used in RT-PCR.

Article Snippet: The mAb Col-1 was purchased from Invitrogen (Carlsbad, CA) and the anti-human CEACAM1 mAb clone 283340 from R&D systems (Minneapolis, MN).

Techniques:

Journal: Scientific Reports

Article Title: CEACAM1 promotes melanoma metastasis and is involved in the regulation of the EMT associated gene network in melanoma cells

doi: 10.1038/s41598-018-30338-4

Figure Lengend Snippet: CEACAM1 knockdown in human melanoma cells and in vitro characterization. ( A ) Left Panel: Flow Cytometry, Histogram. Staining of MeWo CEACAM1 kd (orange curve, respective isotype control in grey) and MeWo Luc (teal, respective isotype control in black) for CEACAM1. Right Panel: In contrast to MeWo Luc, surface CEACAM1 is reduced by over 90% in MeWo CEACAM1 kd, given is the percentage of surface CEACAM1 on MeWo CEACAM kd cells as determined by flow cytometry (Staining and measurement repeated twice; respective MeWo Luc signals set as 100%). ( B ) In vitro XTT proliferation assay with MeWo CEACAM1 kd and MeWo Luc. A slight, but significant increase (p = 0.014) in proliferation was observed for MeWo CEACAM1 kd compared with the MeWo Luc controls. The experiment was repeated twice with 6 replicates (wells) each time (n = 18 each group). ( C ) In vitro migration in a transwell assay with MeWo Luc and MeWo CEACAM1 kd: Significant difference in migration between the two cell lines was observed (p = 0.046). The experiment was repeated once with 3 replicates (wells) each time (n = 6 for each group).

Article Snippet: Briefly, melanoma cells were stained with mouse anti-human PE-labeled CEACAM1/CD66a (R&D, Wiesbaden, Germany), PE-labeled anti-CEACAM3 (Sino Biological, Beijing P.R.

Techniques: In Vitro, Flow Cytometry, Staining, Proliferation Assay, Migration, Transwell Assay

Journal: Scientific Reports

Article Title: CEACAM1 promotes melanoma metastasis and is involved in the regulation of the EMT associated gene network in melanoma cells

doi: 10.1038/s41598-018-30338-4

Figure Lengend Snippet: Spontaneous metastasis xenograft model of human melanoma. ( A ) Kaplan-Meier overall-survival analysis (OS) for scid mice with subcutaneously injected MeWo cells revealed significantly prolonged OS in mice inoculated with MeWo CEACAM1 kd 1 against MeWo Luc inoculated animals (p < 0.001). ( B ) Tumor weight at the time of death was not significantly different between the two groups (p = 0.164; n = 11 Luc group and n = 12 CEACAM kd group). ( C ) Immunohistochemical staining of paraffin embedded tumor tissue (xenograft MeWo tumors in scid mice) for CEACAM1 demonstrated the stability of the CEACAM1 knockdown in the subcutaneous tumors: High CEACAM1 was detected in the MeWo Luc tumors (left panel) whereas little to no CEACAM1 (in red) could be shown in the MeWo CEACAM1 kd tumors (right panel). scale bar: 100 µm. ( D ) Knockdown of CEACAM1 significantly decreases metastasis (adj. for survival time and tumor weight; p = 0.016; n = 11 Luc group and n = 10 CEACAM kd group). ( E ) CEACAM1 knockdown does not significantly alter the number of circulating tumor cells in the blood of scid mice (p = 0.756, n = 10 each group). Quantification of circulating (human) tumor cells in the blood of scid mice inoculated with MeWo CEACAM1 kd (orange) compared with controls (MeWo Luc, teal) by quantitative real-time PCR.

Article Snippet: Briefly, melanoma cells were stained with mouse anti-human PE-labeled CEACAM1/CD66a (R&D, Wiesbaden, Germany), PE-labeled anti-CEACAM3 (Sino Biological, Beijing P.R.

Techniques: Injection, Immunohistochemical staining, Staining, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: CEACAM1 promotes melanoma metastasis and is involved in the regulation of the EMT associated gene network in melanoma cells

doi: 10.1038/s41598-018-30338-4

Figure Lengend Snippet: IGFBP7 expression is up- and Latexin expression is downregulated in the xenograft melanoma induced by CEACAM1 kd cells. ( A ) IGFBP7 and Latexin expression are significantly regulated on the transcriptional level as determined by qRT-PCR with template cDNA derived from RNA from MeWo tumors (human melanoma cell line) subcutaneously grown in scid mice. Please note that lower Cp values mean higher levels of expression: IGFBP7 expression is significantly higher in MeWo CEACAM1 kd than in MeWo Luc control tumors whereas latexin expression is significantly lower in the MeWo CEACAM1 kd tumors. Each experiment was performed twice with 5 replicates each time. ( B ) Immunohistochemical staining of paraffin embedded tumor tissue (xenograft MeWo tumors in scid mice) for IGFBP7 and latexin demonstrated respective up- and downregulation of these two proteins. Little to no IGFBP7 (in red) could be shown in the MeWo Luc tumors (upper left panel) whereas high IGFBP7 was detected in the MeWo CEACAM1 kd tumors (lower left panel). Vice versa, high latexin expression could be detected in the MeWo Luc tumors (upper right panel) and little to no latexin expression in the CEACAM1 kd tumors (lower right panel). ( C ) IGFBP7 and latexin expression are up- and downregulated, respectively, on the cell culture level in MeWo CEACAM1 kd cells compared with MeWo Luc cells: Flow cytometric determination of intracellular IGFBP7 and latexin in MeWo Luc (teal curves) and MeWo CEACAM1 kd cells (orange curves) with corresponding isotype controls (black curves).

Article Snippet: Briefly, melanoma cells were stained with mouse anti-human PE-labeled CEACAM1/CD66a (R&D, Wiesbaden, Germany), PE-labeled anti-CEACAM3 (Sino Biological, Beijing P.R.

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Immunohistochemical staining, Staining, Cell Culture

Journal: Scientific Reports

Article Title: CEACAM1 promotes melanoma metastasis and is involved in the regulation of the EMT associated gene network in melanoma cells

doi: 10.1038/s41598-018-30338-4

Figure Lengend Snippet: CEACAM1 expression in melanoma is inversely correlated with patient survival. ( A ) Kaplan-Meier overall-survival analysis (OS): CEACAM1 expression in melanoma samples was visualized by CEACAM1 specific immunohistochemistry in melanoma samples from 113 patients. Patients were grouped as CEACM1 “no/low” staining or CEACAM1 “medium/high” staining. CEACAM “no/low” patients show a significantly longer overall survival than CEACAM “medium/high” patients (p < 0.001). ( B ) A melanoma with high CEACAM1 expression: Immunohistochemical staining of paraffin embedded patient tissue for CEACAM1. ( C ) A CEACAM1 negative melanoma: Immunohistochemical staining of paraffin embedded patient tissue for CEACAM1.

Article Snippet: Briefly, melanoma cells were stained with mouse anti-human PE-labeled CEACAM1/CD66a (R&D, Wiesbaden, Germany), PE-labeled anti-CEACAM3 (Sino Biological, Beijing P.R.

Techniques: Expressing, Immunohistochemistry, Staining, Immunohistochemical staining

Journal: Aging Cell

Article Title: Aging‐related carcinoembryonic antigen‐related cell adhesion molecule 1 signaling promotes vascular dysfunction

doi: 10.1111/acel.13025

Figure Lengend Snippet: Expression of carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) and VEGF is upregulated in aging human vasculature. (a) Human internal thoracic artery ( HITA ) specimens were grouped according to age in three groups: <60, 60–70 and >70 years. Immunohistochemical analyses demonstrated an age‐dependent upregulation of CEACAM 1 and VEGF , especially within the intima. (b) Similar results were obtained by immunoblotting. The transcription factor HIF ‐1α that is involved in VEGF gene expression was also upregulated with advanced age. A, adventitia; I, intima; M, media

Article Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 protein expression was knocked down in endothelial EA.hy926 cells using a siRNA specific for human Cc1 (CEACAM1 siRNA (h); Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Western Blot, Gene Expression

Journal: Aging Cell

Article Title: Aging‐related carcinoembryonic antigen‐related cell adhesion molecule 1 signaling promotes vascular dysfunction

doi: 10.1111/acel.13025

Figure Lengend Snippet: Expression of carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1), VEGF , and VEGFR ‐2 is age‐dependently upregulated in murine aortae. (a) Immunohistochemical staining of aortic cross sections derived from 2‐ to 9‐month‐old mice. Aortae of 9‐month‐old mice showed a distinct upregulation of CEACAM 1, VEGF , and VEGFR ‐2 compared to aortae of 2‐month‐old mice. (b) Quantification of CEACAM 1(+), VEGF (+), and VEGFR ‐2(+) cells within cross sections of 2‐ and 9‐month‐old mice. Cell count was normalized to the inner aortic circumference. For all three proteins, the number of positive cells increased with age. (c) Immunohistochemical analyses of aortic cross sections of 9‐month‐old mice. Especially within the intima and adventitia, a high number of cells co‐expressing CEACAM 1 with VEGF and VEGFR ‐2, respectively, were present. A, adventitia; I, intima; L, lumen; M, media. * p < 0.05

Article Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 protein expression was knocked down in endothelial EA.hy926 cells using a siRNA specific for human Cc1 (CEACAM1 siRNA (h); Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Staining, Derivative Assay, Cell Counting

Journal: Aging Cell

Article Title: Aging‐related carcinoembryonic antigen‐related cell adhesion molecule 1 signaling promotes vascular dysfunction

doi: 10.1111/acel.13025

Figure Lengend Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) promotes aortic expression of VEGF and VEGFR ‐2 in 9‐month‐old mice. (a) CEACAM 1 deficiency resulted in a reduced expression of VEGF as indicated by immunohistochemical analyses of murine aortic cross sections and immunoblotting, respectively. (b) Similar to VEGF , expression of VEGFR ‐2 was lower in aortae of Cc1 −/− compared to WT mice at the age of 9 months. * p < 0.05

Article Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 protein expression was knocked down in endothelial EA.hy926 cells using a siRNA specific for human Cc1 (CEACAM1 siRNA (h); Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Western Blot

Journal: Aging Cell

Article Title: Aging‐related carcinoembryonic antigen‐related cell adhesion molecule 1 signaling promotes vascular dysfunction

doi: 10.1111/acel.13025

Figure Lengend Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) promotes age‐dependent upregulation of TNF ‐α and oxidative stress. (a) Immunohistochemical analyses of aortic cross sections and immunoblotting demonstrated that CEACAM 1 promotes age‐dependent upregulation of TNF ‐α in 9‐month‐old mice. (b) Vice versa, TNF ‐α (50 ng/ml, 72 hr) upregulated expression of CEACAM 1 in cultured endothelial EA .hy926 cells. This TNF ‐α‐mediated upregulation of CEACAM 1 was partially attenuated by concomitant application of the selective NF ‐κB inhibitor Bay 11‐7085 (10 μM, 72 hr). (c) Aortic mRNA expression of the pro‐oxidative NADPH oxidase subunit NOX 2 was significantly higher in 9‐month‐old WT mice when compared to age‐matched Cc1 −/− mice. (d) 4‐ HNE , a marker of oxidative stress‐mediated lipid peroxidation accumulated in 9‐month‐old WT mice, but not in Cc1 −/− mice, as demonstrated by immunohistochemical analyses of aortic cross sections. (e) Compared to Cc1 −/− mice, the presence of the transcription factor HIF ‐1α was increased in aortae of 9‐month‐old WT mice. * p < 0.05

Article Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 protein expression was knocked down in endothelial EA.hy926 cells using a siRNA specific for human Cc1 (CEACAM1 siRNA (h); Santa Cruz Biotechnology).

Techniques: Immunohistochemical staining, Western Blot, Expressing, Cell Culture, Marker

Journal: Aging Cell

Article Title: Aging‐related carcinoembryonic antigen‐related cell adhesion molecule 1 signaling promotes vascular dysfunction

doi: 10.1111/acel.13025

Figure Lengend Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) promotes age‐dependent vascular hyperpermeability and aortic collagen accumulation. (a) Aortic cross sections of 9‐month‐old WT and Cc1 −/− mice after in situ perfusion with an Evans Blue‐containing solution. Aortic subendothelial dye deposition was higher in WT mice indicating increased endothelial permeability compared to Cc1 −/− mice. (b) Detection of collagen by picrosirius red staining of aortic cross sections from 9‐month‐old WT and Cc1 −/− mice. In the presence of CEACAM 1, there was a distinct age‐related accumulation of collagen fibrils, especially within the aortic media. (c) Immunohistochemical analyses of the expression of TGF ‐β and TGF ‐βR1 in aortic cross sections of 9‐month‐old WT and Cc1 −/− mice. In WT mice, both proteins were expressed higher suggesting augmented pro‐fibrotic TGF ‐β/ TGF ‐βR1 signaling in the presence of CEACAM 1. * p < 0.05

Article Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 protein expression was knocked down in endothelial EA.hy926 cells using a siRNA specific for human Cc1 (CEACAM1 siRNA (h); Santa Cruz Biotechnology).

Techniques: In Situ, Permeability, Staining, Immunohistochemical staining, Expressing

Journal: Aging Cell

Article Title: Aging‐related carcinoembryonic antigen‐related cell adhesion molecule 1 signaling promotes vascular dysfunction

doi: 10.1111/acel.13025

Figure Lengend Snippet: Knockdown of carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) in endothelial cells in vitro mimics in vivo effects of CEACAM 1 deficiency on fibrosis‐, oxidative stress‐, and inflammation‐related genes. (a) Immunoblot detection of CEACAM 1 protein in control si RNA or Cc1 si RNA ‐transfected EA .hy926 cells after different time points and upon TNF ‐α application. CEACAM 1 protein was reduced by approximately 50% 2–4 days after Cc1 si RNA transfection. TNF ‐α (50 ng/ml, 72 hr) induced a huge upregulation of CEACAM 1 protein in control si RNA ‐transfected cells. However, even in the presence of TNF ‐α, Cc1 si RNA efficiently knocked down CEACAM 1 protein. (b) Analysis of mRNA expression under basal conditions 4 days after control and Cc1 si RNA transfection, respectively. Expression of TGF ‐β, TGF ‐βR1, and NOX 4 was significantly higher due to CEACAM 1 knockdown, whereas TNFR 1 was not affected. TNF ‐α was not detectable (n.d.) under basal conditions. (c) Analysis of mRNA expression 4 days after control and Cc1 si RNA transfection, respectively. One day after transfection, both groups of cells were stimulated with TNF ‐α (50 ng/ml) for the remaining 72 hr. The differences in the expression of TGF ‐β, TGF ‐βR1, and NOX 4 between control and Cc1 si RNA ‐transfected cells seen under basal conditions were equalized by TNF ‐α. Again, expression of TNFR 1 was unaffected by the treatment. However, TNF ‐α induced its own expression in both groups of cells. This induction was much more pronounced in control si RNA ‐transfected cells. * p < 0.05

Article Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 protein expression was knocked down in endothelial EA.hy926 cells using a siRNA specific for human Cc1 (CEACAM1 siRNA (h); Santa Cruz Biotechnology).

Techniques: Knockdown, In Vitro, In Vivo, Western Blot, Control, Transfection, Expressing

Journal: Aging Cell

Article Title: Aging‐related carcinoembryonic antigen‐related cell adhesion molecule 1 signaling promotes vascular dysfunction

doi: 10.1111/acel.13025

Figure Lengend Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) as a central mediator of vascular aging. In the course of vascular aging, CEACAM 1 expression is increased. CEACAM 1 promotes aortic upregulation of the pro‐inflammatory cytokine TNF ‐α that, in turn, further contributes to an enhanced CEACAM 1 expression. This mutual influence establishes a feed‐forward loop maintaining a chronic pro‐inflammatory milieu within the vessel wall. Upregulated TNF ‐α may induce expression of pro‐oxidative enzymes, that is, NOX 2, that contribute to an oxidative stress‐mediated stabilization of HIF ‐1α. HIF ‐1α that induces VEGF / VEGFR ‐2 signaling thus promotes endothelial hyperpermeability of aged vessels. Beside this pathway leading to age‐related endothelial dysfunction, upregulated TNF ‐α may also account for the age‐dependently increased arterial fibrosis via enhanced TGF ‐β/ TGF ‐βR1 signaling. In the presence of additional risk factors, these CEACAM 1‐mediated age‐dependent vascular alterations may transform into the development of vascular disorders, that is, atherosclerosis

Article Snippet: Carcinoembryonic antigen‐related cell adhesion molecule 1 protein expression was knocked down in endothelial EA.hy926 cells using a siRNA specific for human Cc1 (CEACAM1 siRNA (h); Santa Cruz Biotechnology).

Techniques: Expressing