Journal: Nature Communications

Article Title: An immune checkpoint score system for prognostic evaluation and adjuvant chemotherapy selection in gastric cancer

doi: 10.1038/s41467-020-20260-7

Figure Lengend Snippet: Multi-colour immunofluorescence staining of anti-CKpan and CD45 antibodies with CD155, NECTIN2, CEACAM1, HMGB1, SIGLEC6 or CD44 antibodies. Scale bar = 200 μm.

Article Snippet: Based on the collection and review of relevant literature (Supplementary Table ), we selected 20 immune checkpoints for immunohistochemical staining analysis: CD73 (ab175396, Abcam, 1 : 200), Galectin-9 (54330 S, Cell Signaling Technology (CST), 1 : 800), HMGB1 (ab18256, Abcam, 1 : 1000), FAS-L (ab186671, Abcam, 1 : 200), SIGLEC6 (ab38581, Abcam, 1 : 200), SIGLEC15 (ab174723, Abcam, 1 : 200), TLR4 (ab13556, Abcam, 1 : 200), ADENOSINE (ab40002, Abcam, 1 : 250), CEACAM1 (44464S, CST, 1 : 400), NECTIN2 (95333S, CST, 1 : 200), CD44 (3570S, CST, 1 : 50), CD155 (81254 S, CST, 1 : 200), VISTA (54979S, CST.

Techniques: Immunofluorescence, Staining

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a Left panel. Log 2 fold-change of sgRNA counts between days 14 and 0 ( P < 0.01; two-sided permutation test) are plotted against the Z ratio of mRNA expression between MCL and naïve-B cells ( P < 0.05; two-sided t -test). Right panel. Overlap of selected genes depleted at least twofold and overexpressed in MCL with pan-dependent genes and genes associated with poor prognosis . Numbers in green boxes indicate filtering strategy (see text). b Shown are mean values of selected normalized sgRNA counts on days 0 and 14 from two biological replicates. c Immunoblots show CEACAM1 knockout in JEKO-1 cells transduced with control (gNTC) or CEACAM1 gRNAs (gCC1) followed by anti-IgM antibody stimulation (2 μg/ml, 5 min). d JEKO-1 cells were transduced with indicated sgRNAs, and viable, GFP + -transduced cells were monitored over time by FACS. Shown are the means of GFP+ fractions compared to day-2 samples from three biological replicates. Error bars, SD. * P < 0.05 by a two-sided, paired t -test. e Immunoblots show CEACAM1 knockdown by shRNA in JEKO-1 cells. f Indicated cells were transduced with CEACAM1 shRNA and viable, propidium iodide (PI)-negative cells were assessed by FACS over time. Shown are the means of PI-negative fractions compared to day-2 samples from three biological replicates. Error bars, SD. * P < 0.05, ** P < 0.01 by a two-sided, paired t -test. g CEACAM1 is required for MCL survival in vivo. JEKO-1 cells were transduced with control or CEACAM1 shRNA and intravenously transplanted into NSG mice. Shown are weekly bioluminescence images of six mice/group. h Line graphs show the means of bioluminescence signals measuring tumor growth in mice described in ( g ). Error bars, SEM. * P < 0.05 by a one-sided t -test. i Kaplan–Meier survival analysis of mice shown in ( g ). P value, log - rank test. j Top panel, Generation of double SOX11/CCND1 transgenic (DT) and CEACAM1-deficient mice. Middle panel, Representative FACS plots show MCL-like population (CD19 + CD5 + CD23-). Bottom panel, bar graphs show means of %MCL-like cells from the peripheral blood of indicated mice and sample sizes. Error bars, SD. P values, two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: For 10 s and cleared by centrifugation at 14,000 × g for 15 min at 4 o C. One percent of the lysate (~10 μl) was saved as input and the rest was incubated with 1 μg of anti-CEACAM1 antibody (Santa Cruz Biotechnology, TX, USA) or isotype control antibody (SouthernBiotech, AL, USA) together with 30 μl of protein G Sepharose beads (Pierce Biotechnology, MA, USA) by rotation at 4 o C for 8 h. Antibody-bound beads were washed six times in 1 ml of IP buffer by centrifugation at 8000 × g for 1 min at 4 o C. Washed Sepharose beads were resuspended in 1x sample buffer containing 5% beta-mercaptoethanol, heat-denatured for 5 min at 95 o C, and analyzed by SDS-PAGE and immunoblotting.

Techniques: Expressing, Western Blot, Knock-Out, Transduction, Control, Knockdown, shRNA, In Vivo, Transgenic Assay

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a – c Box plots show CEACAM1 mRNA expression levels in MCL and other cell types from the datasets GSE2350 ( a , b ) and GSE132929 ( c ). The boxes extend from the 25th to 75th percentiles with the center line as median and whiskers drawn from 10th to 90th percentiles. **** P < 0.0001 by a two-sided Mann–Whitney U - test. GC germinal center, Mem memory, Fo follicular, BL Burkitt lymphoma, DLBCL diffuse large B-cell lymphoma, FL follicular lymphoma, PEL primary effusion lymphoma. d Representative immunoblot analysis of CEACAM1 expression in indicated cells from at least three independent experiments. Numbers below bands represent densitometric values of CEACAM1 signals normalized over GAPDH loading controls. e Flow cytom e try analysis of surface CEACAM1 expression in MCL PDXs compared to CEACAM1-negative RAMOS cells from at least two independent experiments. Live cell gating in this experiment and throughout the study is described in Supplementary Fig. . f Representative immunohistochemistry images showing varying CEACAM1 staining levels for indicated formalin-fixed paraffin-embedded tissue microarrays from three independent experiments. In cHL tissue, background plasma cells are also stained positive. cHL classic Hodgkin lymphoma, CLL chronic lymphocytic leukemia. See Table for a summary of CEACAM1 positivity in specific diseases. Source data are provided as a Source Data file.

Article Snippet: For 10 s and cleared by centrifugation at 14,000 × g for 15 min at 4 o C. One percent of the lysate (~10 μl) was saved as input and the rest was incubated with 1 μg of anti-CEACAM1 antibody (Santa Cruz Biotechnology, TX, USA) or isotype control antibody (SouthernBiotech, AL, USA) together with 30 μl of protein G Sepharose beads (Pierce Biotechnology, MA, USA) by rotation at 4 o C for 8 h. Antibody-bound beads were washed six times in 1 ml of IP buffer by centrifugation at 8000 × g for 1 min at 4 o C. Washed Sepharose beads were resuspended in 1x sample buffer containing 5% beta-mercaptoethanol, heat-denatured for 5 min at 95 o C, and analyzed by SDS-PAGE and immunoblotting.

Techniques: Expressing, MANN-WHITNEY, Western Blot, Immunohistochemistry, Staining, Formalin-fixed Paraffin-Embedded, Clinical Proteomics

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a – e Indicated cell lines were transduced with control or CEACAM1 shRNA, followed by stimulation with anti-IgM (1 μg/mL). a Shown are normalized Ca 2+ signals. **** P < 0.0001 by two-way ANOVA. Data were representative of at least three independent experiments. b , c Immunoblots show effects of CEACAM1 knockdown on BCR signaling components. Data were representative of three independent experiments. d , e Immunoblots show CEACAM1 knockdown (top panels) and its effect on Ca 2+ signals (bottom panels). f Top panel, immunoblots of splenocytes from wild type (+/+) or Ceacam1-deficient (−/−) mouse with indicated antibodies. Bottom panel, Ca 2+ signals of indicated splenic B220 + B cells. Data from ( d – f ) are representative of at least two independent experiments. g CEACAM1 mRNA expression levels in CD19+ sorted MCL cells from peripheral blood (PB) or lymph nodes (LN) analyzed from the GSE70910 dataset . Closed circles or squares represent individual patient samples. Horizontal bars from each group indicate mean mRNA expression. P value is from a two-sided unpaired t -test with Welch’s correction. h CEACAM1 expression is correlated with ibrutinib response. Left panels, FACS plots showing surface CEACAM1 expression on indicated MCL and MZL cell lines. Isotype, negative isotype antibody on Z-138 cells. Right panels, Indicated cell lines were treated with indicated doses of ibrutinib for 4 days, and viable propidium iodide (PI)-negative cells were assessed by flow cytometry. Line graphs showing means of normalized PI-negative fractions from three independent experiments. Error bars, SD. Fifty-percent inhibition concentration (IC 50 ) values were calculated by GraphPad Prism v8. i , j ITIM tyrosine residues are required for BCR signaling. JEKO-1 cells transduced with control (gNTC) or CEACAM1 gRNA (gCC1), followed by reintroducing WT CEACAM1 (4 L), CC1-4L-Y493F/Y520F mutant (YY/FF), or short cytoplasmic tail (4S). Controls or reconstituted cell lines were verified for CEACAM1 expression by FACS using B1.1 antibody ( i ) and stimulated with 1 μg/mL of anti-IgM for 5 min, followed by immunoblotting with indicated antibodies, including the CEACAM1 E1 antibody ( j ). Data from ( i , j ) are representative of at least two independent experiments. Source data are provided as a Source Data file.

Article Snippet: For 10 s and cleared by centrifugation at 14,000 × g for 15 min at 4 o C. One percent of the lysate (~10 μl) was saved as input and the rest was incubated with 1 μg of anti-CEACAM1 antibody (Santa Cruz Biotechnology, TX, USA) or isotype control antibody (SouthernBiotech, AL, USA) together with 30 μl of protein G Sepharose beads (Pierce Biotechnology, MA, USA) by rotation at 4 o C for 8 h. Antibody-bound beads were washed six times in 1 ml of IP buffer by centrifugation at 8000 × g for 1 min at 4 o C. Washed Sepharose beads were resuspended in 1x sample buffer containing 5% beta-mercaptoethanol, heat-denatured for 5 min at 95 o C, and analyzed by SDS-PAGE and immunoblotting.

Techniques: Transduction, Control, shRNA, Western Blot, Knockdown, Expressing, Flow Cytometry, Inhibition, Concentration Assay, Mutagenesis

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a Diagram of sucrose-density gradient fractionation of lipid rafts. b Left panels, Immunoblots of fractions indicated in ( a ) from control or CEACAM1-knockout JEKO-1 cells stimulated with control or anti-IgM antibody (1 μg/ml) for 2 min. Right panels, immunoblot signal quantification of fraction I (red dashed-line box) after normalization to Flotillin-1 and unstimulated controls. Shown are the means of fold changes from three independent experiments. Error bars, SD. ** P < 0.01, * P < 0.05 by a two-sided, paired t -test. ns not significant. c Left panels, Control (gNTC) or CEACAM1 knockout (gCC1) JEKO-1 cells were stimulated with 2 μg/ml anti-IgM antibody for 2 min. Shown are representative cells from confocal immunofluorescence images in Supplementary Fig. of control and IgM-stimulated cells co-stained with anti-FLNA (green) and anti-LYN (red) antibodies, followed by nuclear staining with DAPI (blue). Scale bar, 2 μm. Right panels, quantified fluorescent signals for each cell from the samples described. Shown are the sums of intensities in signal-positive areas per cell in arbitrary units from three independent experiments. Horizontal red bars indicate the mean. Approximately 200 cells from each sample were analyzed. **** P < 0.0001 by a two-sided unpaired t -test. ns not significant. d CEACAM1 is required for lipid-raft assembly. Top panels, Control, CEACAM1 knockout (CC1 KO), or CC1 KO + CEACAM1-4L (4 L) JEKO-1 cells were stimulated with 2 μg/ml anti-IgM antibody for 5 min. Shown are representative confocal immunofluorescence images of control and IgM-stimulated cells stained with anti-CEACAM1 antibody (green), GM1 via cholera toxin B (red), p-SRC Y416 antibody (green), or F-actin via Actin-Stain 555 Phalloidin (red) followed by nuclear staining with DAPI (blue). Scale bar, 10 μm. Bottom panels, quantified fluorescent signals from the samples described in the top panels. Shown are the sums of intensities in signal-positive areas per cell in arbitrary units from three independent experiments. Horizontal red bars indicate the mean. Approximately 200 cells from each sample were analyzed. **** P < 0.0001 by a two-sided unpaired t -test. Source data are provided as a Source Data file.

Article Snippet: For 10 s and cleared by centrifugation at 14,000 × g for 15 min at 4 o C. One percent of the lysate (~10 μl) was saved as input and the rest was incubated with 1 μg of anti-CEACAM1 antibody (Santa Cruz Biotechnology, TX, USA) or isotype control antibody (SouthernBiotech, AL, USA) together with 30 μl of protein G Sepharose beads (Pierce Biotechnology, MA, USA) by rotation at 4 o C for 8 h. Antibody-bound beads were washed six times in 1 ml of IP buffer by centrifugation at 8000 × g for 1 min at 4 o C. Washed Sepharose beads were resuspended in 1x sample buffer containing 5% beta-mercaptoethanol, heat-denatured for 5 min at 95 o C, and analyzed by SDS-PAGE and immunoblotting.

Techniques: Fractionation, Western Blot, Control, Knock-Out, Immunofluorescence, Staining

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a , b Top panels, Proximity ligation assay (PLA) showing interactions (visualized as red dots using Airyscan FAST 2D confocal microscope and a 40x/1.2NA water objective) between CEACAM1 (CC1) and the indicated proteins in JEKO-1 and MINO cells stimulated with 2 μg/ml of anti-IgM antibody for the indicated times. Bottom panels, Quantification of PLA signals shown in the top panels for ~200 cells on average from three independent experiments using QuPath 0.3.2 software. **** P < 0.0001, *** P < 0.001 by a two-tailed unpaired t -test. ns not significant. c , d Immunoprecipitation analysis of CEACAM1 interactions. Top panels, JEKO-1 or MINO cells were stimulated with 2 μg/ml of anti-IgM antibody for the indicated times, and CEACAM1 was immunoprecipitated with a CEACAM1-specific antibody or IgG control antibodies, followed by immunoblotting with the indicated antibodies. One percent of the total lysates was used as an input control. Bottom panels, Quantification of indicated co-IP signals shown in the top panels. Bar graphs show the means and individual densitometric values from three independent experiments for each timepoint normalized to the CEACAM1 pull-down signals. Error bars, SD. *** P < 0.001, ** P < 0.01, * P < 0.05 by a two-sided unpaired t -test. ns not significant. Source data are provided as a Source Data file.

Article Snippet: For 10 s and cleared by centrifugation at 14,000 × g for 15 min at 4 o C. One percent of the lysate (~10 μl) was saved as input and the rest was incubated with 1 μg of anti-CEACAM1 antibody (Santa Cruz Biotechnology, TX, USA) or isotype control antibody (SouthernBiotech, AL, USA) together with 30 μl of protein G Sepharose beads (Pierce Biotechnology, MA, USA) by rotation at 4 o C for 8 h. Antibody-bound beads were washed six times in 1 ml of IP buffer by centrifugation at 8000 × g for 1 min at 4 o C. Washed Sepharose beads were resuspended in 1x sample buffer containing 5% beta-mercaptoethanol, heat-denatured for 5 min at 95 o C, and analyzed by SDS-PAGE and immunoblotting.

Techniques: Proximity Ligation Assay, Microscopy, Software, Two Tailed Test, Immunoprecipitation, Control, Western Blot, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a Left panel, Immunoprecipitation analysis of CEACAM1 interactions. CEACAM1-knockout JEKO-1 cells were transduced with either full-length (4 L) or N-domain truncated (ΔN) CEACAM1 constructs, stimulated with 2 μg/ml anti-IgM antibody for the indicated times, followed by CEACAM1 immunoprecipitation and immunoblotting with the indicated antibodies. One percent of the total lysates was used as an input control. p-Y, anti-phosphotyrosine antibody clone 4G10. Right panels, Quantification of indicated co-IP signals shown in the left panels. Bar graphs show the means and individual densitometric values from four independent experiments for each timepoint normalized to the CEACAM1 pull-down signals. Error bars, SD. ** P < 0.01, * P < 0.05 by two-way ANOVA. b Proximity ligation assay (PLA) showing interactions (visualized as red dots using Airyscan FAST 2D confocal microscope and a 40x/1.2NA water objective) between 4 L or ΔN and the indicated proteins. c Quantitation of PLA signals shown in ( b ) for 100–300 cells on average from three independent experiments using QuPath 0.3.2 software. Error bars indicate means with S.D. **** P < 0.0001 by two-sided Mann–Whitney U -test. ns not significant. Source data are provided as a Source Data file.

Article Snippet: For 10 s and cleared by centrifugation at 14,000 × g for 15 min at 4 o C. One percent of the lysate (~10 μl) was saved as input and the rest was incubated with 1 μg of anti-CEACAM1 antibody (Santa Cruz Biotechnology, TX, USA) or isotype control antibody (SouthernBiotech, AL, USA) together with 30 μl of protein G Sepharose beads (Pierce Biotechnology, MA, USA) by rotation at 4 o C for 8 h. Antibody-bound beads were washed six times in 1 ml of IP buffer by centrifugation at 8000 × g for 1 min at 4 o C. Washed Sepharose beads were resuspended in 1x sample buffer containing 5% beta-mercaptoethanol, heat-denatured for 5 min at 95 o C, and analyzed by SDS-PAGE and immunoblotting.

Techniques: Immunoprecipitation, Knock-Out, Transduction, Construct, Western Blot, Control, Co-Immunoprecipitation Assay, Proximity Ligation Assay, Microscopy, Quantitation Assay, Software, MANN-WHITNEY

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: a Left panel, CEACAM1 suppresses BCR signaling in Z-138 cells. Z-138 cells were transduced with control empty vector (EV), WT CEACAM1 (CC1-4L), or short cytoplasmic tail CEACAM1 (CC1-4S). Transduced cells were stimulated with 2 μg/mL of anti-IgM F(ab’)2 fragments for the indicated times, followed by immunoblot analysis probed with the indicated antibodies. HA, hemagglutinin, a protein tag in-framed with CEACAM1 to detect the 4S isoform. Right panel, Quantification of the immunoblot signals shown in the left panel. Bar graphs show the means of densitometric values from the indicated time points normalized to GAPDH loading controls from two independent experiments. P values, one-sided permutation test. b , c CEACAM1-knockout JEKO-1 cells (gCC1) or Z-138 cells were transduced with either empty vector control or full-length (4 L) CEACAM1 construct, stimulated with 2 μg/ml anti-IgM antibody for the indicated times, followed by CEACAM1 immunoprecipitation and immunoblotting with indicated antibodies. One percent of the total lysates was used as an input control ( b ). The samples shown in ( b ) derive from the same experiment, but different gels for CEACAM1, p-SYK Y352 , SYK, SHP-1, GAPDH, and another for SHP-2 were processed in parallel. Bar graphs show the quantification of the co-IP signals shown in ( c ). Shown are the means of densitometric values from two independent experiments for each timepoint normalized to the CEACAM1 pull-down signals. **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05 by two-way ANOVA. d Left panels, Proximity ligation assay (PLA) showing interactions (visualized as red dots using Airyscan FAST 2D confocal microscope and a 40x/1.2NA water objective) between CEACAM1 (CC1) and the indicated proteins in Z-138 cells or Z-138 cells transduced with CEACAM1-4L followed by stimulation with 2 μg/ml of anti-IgM antibody for the indicated times. Right panels, Quantification of PLA signals shown in the right panels for ~200 cells on average from three independent experiments using QuPath 0.3.2 software. **** P < 0.0001 by a two-sided unpaired t -test with Welch’s correction. Source data are provided as a Source Data file.

Article Snippet: For 10 s and cleared by centrifugation at 14,000 × g for 15 min at 4 o C. One percent of the lysate (~10 μl) was saved as input and the rest was incubated with 1 μg of anti-CEACAM1 antibody (Santa Cruz Biotechnology, TX, USA) or isotype control antibody (SouthernBiotech, AL, USA) together with 30 μl of protein G Sepharose beads (Pierce Biotechnology, MA, USA) by rotation at 4 o C for 8 h. Antibody-bound beads were washed six times in 1 ml of IP buffer by centrifugation at 8000 × g for 1 min at 4 o C. Washed Sepharose beads were resuspended in 1x sample buffer containing 5% beta-mercaptoethanol, heat-denatured for 5 min at 95 o C, and analyzed by SDS-PAGE and immunoblotting.

Techniques: Transduction, Control, Plasmid Preparation, Western Blot, Knock-Out, Construct, Immunoprecipitation, Co-Immunoprecipitation Assay, Proximity Ligation Assay, Microscopy, Software

Journal: Nature Communications

Article Title: CEACAM1 as a mediator of B-cell receptor signaling in mantle cell lymphoma

doi: 10.1038/s41467-025-60208-3

Figure Lengend Snippet: Following antigen stimulation in B-cell lymphomas with abundant CEACAM1 expression, CEACAM1 binds to FLNA, which anchors to the actin cytoskeleton and lipid rafts, and recruits SYK to the proximity of CD79A/B to enhance BCR activity. In cells with low or no CEACAM1 expression (and potentially low SYK expression), SHP-1 and SHP-2 outcompete for CEACAM1 binding, leading to signal attenuation. Created in BioRender. Ngo, V. (2025) https://BioRender.com/dl16wxh .

Article Snippet: For 10 s and cleared by centrifugation at 14,000 × g for 15 min at 4 o C. One percent of the lysate (~10 μl) was saved as input and the rest was incubated with 1 μg of anti-CEACAM1 antibody (Santa Cruz Biotechnology, TX, USA) or isotype control antibody (SouthernBiotech, AL, USA) together with 30 μl of protein G Sepharose beads (Pierce Biotechnology, MA, USA) by rotation at 4 o C for 8 h. Antibody-bound beads were washed six times in 1 ml of IP buffer by centrifugation at 8000 × g for 1 min at 4 o C. Washed Sepharose beads were resuspended in 1x sample buffer containing 5% beta-mercaptoethanol, heat-denatured for 5 min at 95 o C, and analyzed by SDS-PAGE and immunoblotting.

Techniques: Expressing, Activity Assay, Binding Assay

Journal: Oncogene

Article Title: CEA-related cell adhesion molecule-1 is involved in angiogenic switch in prostate cancer.

doi: 10.1038/sj.onc.1209514

Figure Lengend Snippet: Figure 1 (a–h) Expression pattern of carcinoembryonic antigen (CEA)-related cell adhesion molecule-1 (CEACAM1) in normal prostate and prostate cancer tissue. In normal prostate, CEA- CAM1 is visible at the luminal surface of the prostate epithelium using the antibody 4D1/C2 (a) but not in the adjacent blood vessels visualized by CD34-staining on a serial section from the same area (b). In high-grade prostate intraepithelial neoplasia (PIN) the epithelial CEACAM1 is almost disappeared while the majority of the adjacent blood vessels (c) exhibits CEACAM1 when compared with the immunostaining for the endothelial marker CD34 on the serial section (d). In Gleason 3 prostate cancer, where tumor cells themselves form glandular structures, CEACAM1 is present at the luminal surface of tumor glands (e) and only a part of the blood vessels exhibit a weak CEACAM1 staining (arrows) (e) as confirmed by CD34 staining on the serial section (f). Also in solid prostate cancer of the stages Gleason 4 þ 5 a part of the tumor vessels exhibits CEACAM1 but there is no CEACAM1 staining in tumor cells (g). In the control section treated only with the secondary antibody no specific staining is visible (h). All sections are counterstained with calcium red. (a–h): 450. The percentage of CEACAM1-positive blood vessels in relation to CD34-positive vessels switches from 13.4% in the normal area with epithelial presence of CEACAM1 to 78.4% in the PIN areas and to 50.8% in the solid tumor area (i) where the epithelial CEACAM1 was disappeared.

Article Snippet: Similar results were obtained using the CEACAM1 antibodies T84.1 and human goat CEACAM1 antibody from R&D Systems (Supplementary Information).

Techniques: Expressing, Staining, Immunostaining, Marker, Control

Journal: Oncogene

Article Title: CEA-related cell adhesion molecule-1 is involved in angiogenic switch in prostate cancer.

doi: 10.1038/sj.onc.1209514

Figure Lengend Snippet: Figure 2 (a–o) Carcinoembryonic antigen (CEA)-related cell adhesion molecule-1 (CEACAM1) overexpression versus silencing in prostate cancer cell line DU-145 and expression of angiogenic factors: quantitative real-time RT–PCR analyses demonstrate the knock down of CEACAM1 at the mRNA level by more than three times in comparison to luciferase silencing used as control (a). CEACAM1 overexpression increases CEACAM1-mRNA by more then 9000 times in comparison to the empty vector transfection used as control (b). Western blot analysis using the CEACAM1-antibody 4D1/C2 shows that the enhanced CEACAM1 protein expression is significantly reduced when DU-145 cells were co-transfected with CEACAM1 expression vector pcDNA3.1/CEACAM1 plus CEACAM1-small interfering ribonucleic acid (siRNA) (c), which shows the specificity and efficiency of the CEACAM1-siRNA. Also the weak basal expression of CEACAM1 at the protein level (empty vector) disappears completely by CEACAM1 silence. Quantitative real-time reverse transcriptase–polymerase chain reaction (RT–PCR) analyses demonstrate that CEACAM1 overexpression suppresses but CEACAM1 silencing increases the expression of potent angiogenic and lymphangiogenic factors such as VEGF-A (d–e), VEGF-C (f–g), VEGF-D (h–i), and the expression of collagen 18 (j–k), the maternal substance of the angiogenesis inhibitor endostatin. In contrast, the expression of Ang1 is increased by CEACAM1 overexpression but significantly decreased by CEACAM1 silencing (l–m). Inversely to Ang1, the expression of Ang2, the naturally occurring antagonist of Ang1, is suppressed by CEACAM1 overexpression, but enhanced by CEACAM1 silencing in DU-145 cells (n–o).

Article Snippet: Similar results were obtained using the CEACAM1 antibodies T84.1 and human goat CEACAM1 antibody from R&D Systems (Supplementary Information).

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Knockdown, Comparison, Luciferase, Control, Plasmid Preparation, Transfection, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Journal: Oncogene

Article Title: CEA-related cell adhesion molecule-1 is involved in angiogenic switch in prostate cancer.

doi: 10.1038/sj.onc.1209514

Figure Lengend Snippet: Figure 3 (a–d) Double immunostaining for carcinoembryonic antigen (CEA)-related cell adhesion molecule-1 (CEACAM1) and vascular endothelial growth factor (VEGF) in human prostate tissue: co-immunostaining for CEACAM1 (red) and VEGF (dark) on normal prostate tissue demonstrate CEACAM1 at the luminal surface (arrowheads) as expected and VEGF staining in the cytoplasm of epithelial cells as well as in the interstitial myocytes (arrows) (a). In prostate glands containing prostate intraepithelial neoplasia (PIN), CEACAM1 is partially downregulated or disappeared completely (arrowheads) but is concurrently upregu- lated in the adjacent blood vessels (arrows) (b). At these sites of CEACAM1 downregulation, a clearly increased VEGF staining is detectable (b). In Gleason 3, CEACAM1 and VEGF are present in a part of the tumor epithelial cells (c) whereas small blood vessels also exhibit CEACAM1 (arrows). Note that in contrast to the normal prostate glands (a) in glands of Gleason 3 CEACAM1 is present not only at the luminal surface but also at the basal-lateral sides of tumor epithelial cells (c). In the higher stages of prostate cancer, as demonstrated here by Gleason 4 þ 5, CEACAM1 is present in the small blood vessels (arrows) but not in tumor cells whereas a strong VEGF staining is found in a part of the tumor cells (d). Pst: prostate stone. 450.

Article Snippet: Similar results were obtained using the CEACAM1 antibodies T84.1 and human goat CEACAM1 antibody from R&D Systems (Supplementary Information).

Techniques: Double Immunostaining, Immunostaining, Staining

Journal: Oncogene

Article Title: CEA-related cell adhesion molecule-1 is involved in angiogenic switch in prostate cancer.

doi: 10.1038/sj.onc.1209514

Figure Lengend Snippet: Figure 5 (a–d) Carcinoembryonic antigen (CEA)-related cell adhesion molecule-1 (CEACAM1) overexpression versus silencing in DU-145 cells and endothelial migration: in comparison to the combined application of VEGF and the supernatant of empty vector-transfected DU-145 cells (a), the simultaneous application of VEGF and the supernatant of CEACAM1-overexpressing DU- 145 cells (b) reduces the endothelial migration in in vitro wound assay by approximately 41%. While the endothelial migration induced by simultaneous application of VEGF and the supernatant of luciferase-silenced DU-145 cells (c) is comparable to that induced by VEGF plus the supernatant of empty vector-transfected DU-145 cells (a) the combined application of VEGF and the supernatant of CEACAM1-silenced DU-145 cells (d) increases the endothelial migration significantly, approximately by more than 41% in comparison to that in (a) and by more than 57% in comparison to that in (b).

Article Snippet: Similar results were obtained using the CEACAM1 antibodies T84.1 and human goat CEACAM1 antibody from R&D Systems (Supplementary Information).

Techniques: Over Expression, Migration, Comparison, Plasmid Preparation, Transfection, In Vitro, Luciferase

Journal: Oncogene

Article Title: CEA-related cell adhesion molecule-1 is involved in angiogenic switch in prostate cancer.

doi: 10.1038/sj.onc.1209514

Figure Lengend Snippet: Figure 6 (a–f) Carcinoembryonic antigen (CEA)-related cell adhesion molecule-1 (CEACAM1) overexpression versus silencing in DU-145 and endothelial tube formation in vitro: endothelial tube (arrows) forming effect of vascular endothelial growth factor (VEGF) (a) is almost abolished when VEGF plus the supernatant of CEACAM1-overexpressing DU-145 cells were applied (b). In contrast, the supernatant of CEACAM1-silenced DU-145 alone induces endothelial tubes (arrows) (c). The measurements of tubular length revealed a 67% increase when VEGF plus the supernatant of CEACAM1-silenced DU-145 were applied simulta- neously (d) in comparison to the application of VEGF alone as showed in panel a. The tube forming (arrows) effect of the combined application of VEGF and the supernatant of luciferase- silenced DU-145 (e) is almost comparable to that of VEGF alone (a). No tubes are visible in the control when endothelial cells were exposed to the basal medium alone (f). 150.

Article Snippet: Similar results were obtained using the CEACAM1 antibodies T84.1 and human goat CEACAM1 antibody from R&D Systems (Supplementary Information).

Techniques: Over Expression, In Vitro, Comparison, Luciferase, Control

Journal: Oncogene

Article Title: CEA-related cell adhesion molecule-1 is involved in angiogenic switch in prostate cancer.

doi: 10.1038/sj.onc.1209514

Figure Lengend Snippet: Figure 7 Inverse role of carcinoembryonic antigen (CEA)-related cell adhesion molecule-1 (CEACAM1) in the regulation of angiogenesis in prostate cancer: the presence of CEACAM1 in the normal prostate epithelium suppresses the expression of potent angiogenic factors such as vascular endothelial growth factor (VEGF)-A, -C, and -D and Ang2 but increases the expression of Ang1, the natural Ang2 antagonist. Via this mechanism CEACAM1 may be involved in the maintenance of vascular stability and integrity, a process probably preventing the angiogenic switch. CEACAM1 downregulation, as it occurs in prostate intraepithelial neoplasia (PIN), is obviously accompanied by an upregulation of angiogenic factors mentioned above but by a downregulation of Ang1. Concurrently, CEACAM1 is upregulated in endothelial cells of angiogenicly activated blood vessels adjacent to PIN. This switch is apparently accompanied by vascular destabilization, a process marking the initiation of angiogenesis. In summary, CEACAM1 seems to play a dual role in the regulation of angiogenesis depending on which cell type is expressing it. While epithelial presence of CEACAM1 functions inverse to the endothelial expression of CEACAM1, the epithelial downregulation but the concurrent endothelial upregulation act synergistically and promote angiogenesis, tumor vascularization, and probably tumor invasion.

Article Snippet: Similar results were obtained using the CEACAM1 antibodies T84.1 and human goat CEACAM1 antibody from R&D Systems (Supplementary Information).

Techniques: Expressing

Journal: iScience

Article Title: CEACAM1 is a direct SOX10 target and inhibits melanoma immune infiltration and stemness.

doi: 10.1016/j.isci.2022.105524

Figure Lengend Snippet: Figure 3. SOX10 expression is correlated with CD8+ T-cell infiltration and CEACAM1 levels (A) In vivo growth comparison between the pBABE control (n = 5) and the pBABE-SOX10 overexpressing (n = 5) YUMM1.7 cell lines in C57 immune-competent mice. (B) Flow cytometry analysis of the CD4+ and CD8+ T-cell populations within the pBABE and pBABE-SOX10 tumors at endpoint. (C) Comparison of tumor size 24 days post injection between pBABE control (n = 4) and the pBABE-SOX10 overexpressing (n = 5) YUMM1.7 cell lines in RAG1 mice. (D) Flow analysis showing the proportion of CEACAM1 positive cells in various YUMM cell lines compared to isotype control. (E) Flow cytometric staining of SOX10 and CEACAM1 in OHRI-13 and SK-MEL28 human melanoma cell lines. Percentage of each group is indicated within their quadrant. (F) Flow cytometric staining of SOX10 and CEACAM1 in the YUMM1.1, YUMM1.7 and YUMM2.1 murine cell lines. Percentage of each group is indicated within their quadrant. Graphs show the mean G SEM. **p < 0.005 by two-tailed t test (B).

Article Snippet: Antibodies SOX10 New England BioLabs Cat# 89356; Clone D5V9L; RRID:AB_2792980 Beta-Actin Sigma-Aldrich Cat# A5316; Clone AC-74; RRID:AB_476743 Histone H3 New England BioLabs Cat# 4620; Clone D2B12; RRID:AB_1904005 Mouse: CEACAM1 BV421 BioLegend Cat# 134531; Clone Mab-CC1; RRID:AB_2687363 Human: CEACAM1 AF647 R&D Systems Cat# FAB2244R; Clone 283340 PDL-1 BV786 BD Biosciences Cat# 741014; Clone MIH5; RRID:AB_2740636

Techniques: Expressing, In Vivo, Comparison, Control, Flow Cytometry, Injection, Staining, Two Tailed Test

Journal: iScience

Article Title: CEACAM1 is a direct SOX10 target and inhibits melanoma immune infiltration and stemness.

doi: 10.1016/j.isci.2022.105524

Figure Lengend Snippet: Figure 4. SOX10 directly activates Ceacam1 on a distal enhancer region (A) YUMM1.1 cells were treated with siRNA at 200nM targeting Sox10 or a non-targeting control for 72 h and Sox10 knockdown was assessed by Immunoblot. (B) 72 h post siRNA transfection cells were isolated and stained for CEACAM1. Representative flow plots were generated with indicated percent CEACAM1+ cells. (C) qPCR was used to compare Sox10 knockdown efficiency with Ceacam1 isoform levels of the cells in (A). (D) YUMM1.7 pBABE and YUMM1.7 pBABE-SOX10 cells were collected and analyzed by qPCR for Sox10 and Ceacam1 isoforms. (E) YUMM1.7 pBABE and YUMM1.7 pBABE-SOX10 cells were isolated and stained for CEACAM1. Representative flow plots were generated with indicated percent CEACAM1+ cells. (F) Luciferase activity measurements from various Ceacam1 promoter fragments in YUMM1.1 and 2.1 cells. (G) RT-PCR of SOX10 ChIP in YUMM1.1 cells from various regions within the CEACAM1 -4135/-2785 fragment showing the fold enrichment over IgG. The red line represents the IgG control set to one. Data is representative of three independent experiments (C, D, F, and G). Graphs show the mean G SEM. *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001 by one-way ANOVA (C and D) or by two-tailed t test (F).

Article Snippet: Antibodies SOX10 New England BioLabs Cat# 89356; Clone D5V9L; RRID:AB_2792980 Beta-Actin Sigma-Aldrich Cat# A5316; Clone AC-74; RRID:AB_476743 Histone H3 New England BioLabs Cat# 4620; Clone D2B12; RRID:AB_1904005 Mouse: CEACAM1 BV421 BioLegend Cat# 134531; Clone Mab-CC1; RRID:AB_2687363 Human: CEACAM1 AF647 R&D Systems Cat# FAB2244R; Clone 283340 PDL-1 BV786 BD Biosciences Cat# 741014; Clone MIH5; RRID:AB_2740636

Techniques: Control, Knockdown, Western Blot, Transfection, Isolation, Staining, Generated, Luciferase, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Two Tailed Test

Journal: iScience

Article Title: CEACAM1 is a direct SOX10 target and inhibits melanoma immune infiltration and stemness.

doi: 10.1016/j.isci.2022.105524

Figure Lengend Snippet: Figure 5. CEACAM1 decreases CD8+ T-Cell infiltration and promotes tumor growth (A) Overlaid flow profiles representing the YUMM1.7 cells stably expressing CEACAM1 Isoform 1 or 4. (B) Quantitation of primary and secondary sphere formation in SOX10-deficient YUMM1.7 cells stably expressing CEACAM1 and controls. (C) Representative images of both primary and secondary spheres from the assays in (B) (Scale bar = 100mM). (D and E) In vivo growth comparison between the pBABE controls (n = 5), the CEACAM1 iso1 (n = 5) and iso4 (n = 4) expressing YUMM1.7 cell lines in NCG immune compromised mice (D) and C57 immune-competent mice (E). (F) Flow cytometry analysis of the CD4+ and CD8+ T-cell populations within the pBABE and pBABE-CEACAM1 tumors at endpoint. Data is representative of three independent experiments (B). Graphs show the mean G SEM. *p < 0.05, **p < 0.005, ***p < 0.001 by one-way ANOVA (B and F).

Article Snippet: Antibodies SOX10 New England BioLabs Cat# 89356; Clone D5V9L; RRID:AB_2792980 Beta-Actin Sigma-Aldrich Cat# A5316; Clone AC-74; RRID:AB_476743 Histone H3 New England BioLabs Cat# 4620; Clone D2B12; RRID:AB_1904005 Mouse: CEACAM1 BV421 BioLegend Cat# 134531; Clone Mab-CC1; RRID:AB_2687363 Human: CEACAM1 AF647 R&D Systems Cat# FAB2244R; Clone 283340 PDL-1 BV786 BD Biosciences Cat# 741014; Clone MIH5; RRID:AB_2740636

Techniques: Stable Transfection, Expressing, Quantitation Assay, In Vivo, Comparison, Flow Cytometry

Journal: iScience

Article Title: CEACAM1 is a direct SOX10 target and inhibits melanoma immune infiltration and stemness.

doi: 10.1016/j.isci.2022.105524

Figure Lengend Snippet: Figure 6. SOX10hi/CEACAM1+ cells show decreased CSC pool and CD8+ T-cell infiltration in tumors (A) Representative flow sorts showing the proportion of CEACAM1+ and CEACAM1-cells obtained following SOX10 expression in YUMM1.7 cells. (B) qPCR analysis for Sox10 and the Ceacam1 isoforms in sorted YUMM1.7 pBABE-SOX10 cells shown in (A). (C) In vivo growth comparison between the pBABE-SOX10 (n = 5), CEACAM1- (n = 5) and CEACAM1+ (n = 5) YUMM1.7 cell lines in C57 immune-competent mice. (D) Flow cytometry analysis of the CD4+ and CD8+ T-cell populations within the pBABE-SOX10, CEACAM1- and CEACAM1+ tumors at endpoint. (E) Quantitation of primary and secondary sphere formation in the YUMM1.7 pBABE-SOX10, CEACAM1- and CEACAM1+ cells. (F) Representative images of both primary and secondary sphere assays from (E) (scale bar = 100mM).

Article Snippet: Antibodies SOX10 New England BioLabs Cat# 89356; Clone D5V9L; RRID:AB_2792980 Beta-Actin Sigma-Aldrich Cat# A5316; Clone AC-74; RRID:AB_476743 Histone H3 New England BioLabs Cat# 4620; Clone D2B12; RRID:AB_1904005 Mouse: CEACAM1 BV421 BioLegend Cat# 134531; Clone Mab-CC1; RRID:AB_2687363 Human: CEACAM1 AF647 R&D Systems Cat# FAB2244R; Clone 283340 PDL-1 BV786 BD Biosciences Cat# 741014; Clone MIH5; RRID:AB_2740636

Techniques: Expressing, In Vivo, Comparison, Flow Cytometry, Quantitation Assay

Journal: Oncogenesis

Article Title: The CEACAM1 tumor suppressor is an ATM and p53-regulated gene required for the induction of cellular senescence by DNA damage

doi: 10.1038/oncsis.2012.7

Figure Lengend Snippet: CEACAM1 is upregulated by NCS in an ATM-dependent manner. ( a ) Parental MCF-10A cells (MCF-10A) or MCF-10A cells stably expressing an ATM shRNA vector (ATM-KD) or a scrambled shRNA vector (CTRL) as a control were incubated for 4 h in the presence or the absence of 20 n M NCS. At the end of the incubation, total RNA was extracted and analyzed for the levels of CEACAM1 or ATM expression by quantitative real-time PCR. The values in the graph represent the mean±s.e.m. from three independent experiments. § P <0.005; # P <0.001. ( b ) MCF-10A cells stably expressing an ATM shRNA vector (ATM-KD) or a scrambled shRNA (CTRL) were incubated for 16 h in the presence or the absence of 5.46 n M NCS. At the end of the incubation, total proteins were extracted and analyzed for the levels of CEACAM1 or Nijmegen breakage syndrome 1 (NBS1) expression by western blotting. ( c ) Parental MCF-10A cells were incubated for 1 h in the presence of 10 μ M KU-55933 or the same dilution of dimethyl sulfoxide (DMSO (solvent)) as a control, followed by 16 h incubation in the presence or the absence of NCS 5.46 n M . At the end of the incubation, total proteins were extracted and analyzed for the levels of CEACAM1 or NBS1 expression by western blotting. In b and c , numbers on the left indicate kDa. ( d ) MCF-10A cells were incubated in the presence of 5.46 n M NCS for the times indicated. At the end of the incubation, the cells were split into two parts and either processed for protein extraction or for total RNA purification. RNAs were analyzed for the levels of CEACAM1, CEACAM1 primary transcript (CEACAM1 int-ex), p21/ Waf1 or BTG2 by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates. Proteins were analyzed for the levels of CEACAM1, p53 Ser 15-p, p53 or β-actin (lower panel). Numbers on the left indicate kDa. ( e ) MCF-10A cells were incubated for 1 h in the presence of 10 μ M KU-55933 or the same volume of DMSO (solvent) as a control, followed by 4 h in the presence of the indicated concentrations of NCS. At the end of the incubation total RNAs were purified and analyzed for the levels of the indicated genes by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates.

Article Snippet: MCF-10A and HCT116 stable transfectants with stable CEACAM1 silencing were established using shRNA constructs against CEACAM1 in pRS vector purchased from OriGene Technologies/Labforce, Nunningen, Switzerland (cat. no. TR314008), according to the manufacturer's instructions.

Techniques: Stable Transfection, Expressing, shRNA, Plasmid Preparation, Control, Incubation, Real-time Polymerase Chain Reaction, Western Blot, Solvent, Protein Extraction, Purification

Journal: Oncogenesis

Article Title: The CEACAM1 tumor suppressor is an ATM and p53-regulated gene required for the induction of cellular senescence by DNA damage

doi: 10.1038/oncsis.2012.7

Figure Lengend Snippet: Induction of CEACAM1 by NCS or X-Rays in MCF-10A, HCT116 or SW48 cells is dependent on p53. ( a ) MCF-10A cells were transfected with p53 siRNA no.3 or a control (CTRL) siRNA as indicated. Two days later, 10 μ M KU-55933 (K) or dimethyl sulphoxide (DMSO) (D) (solvent) was added. One hour later the cells were incubated in the presence or the absence of 20 n M NCS as indicated for an additional 4 h. At the end of the incubation total RNAs were purified and analyzed for the levels of the indicated genes by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates. ( b ) HCT116 or SW48 cells with wild type or inactivated p53 were irradiated with the indicated doses of X-rays and incubated at 37 °C for 6 h. At the end of the incubation total RNAs were purified and analyzed for the levels of the indicated genes by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates. ( c ) MCF-10A cells were incubated for the indicated times in the presence of 10 μ M nutlin-3, or for 6 h in the presence of DMSO (D) (solvent) (left) or for 6 h in the presence of the indicated concentrations of nutlin-3 or the same volume of DMSO (D) (right). At the end of the incubation total RNAs were purified and analyzed for the levels of the indicated genes by quantitative real-time PCR in triplicates. Error bars indicate s.d. within the internal replicates. ( d ) HCT116 cells with wild type or inactivated p53 were incubated in the presence or the absence of NCS 5.46 n M for 16 h in duplicate as indicated. At the end of the incubation the cells were lysed and analysed for the levels of CEACAM1 or NBS1 by western blotting.

Article Snippet: MCF-10A and HCT116 stable transfectants with stable CEACAM1 silencing were established using shRNA constructs against CEACAM1 in pRS vector purchased from OriGene Technologies/Labforce, Nunningen, Switzerland (cat. no. TR314008), according to the manufacturer's instructions.

Techniques: Transfection, Control, Solvent, Incubation, Purification, Real-time Polymerase Chain Reaction, Irradiation, Western Blot

Journal: Oncogenesis

Article Title: The CEACAM1 tumor suppressor is an ATM and p53-regulated gene required for the induction of cellular senescence by DNA damage

doi: 10.1038/oncsis.2012.7

Figure Lengend Snippet: Establishment and phenotype of MCF-10A cells or HCT116 cells with stable silencing of CEACAM1. ( a ) HCT116 cells stably transfected with CEACAM1 shRNA vector no. 26 (CEACAM1-KD 026), with CEACAM1 shRNA vector no. 28 (CEACAM1-KD 028) or with a control shRNA vector (CTRL) were lysed and analyzed for CEACAM1 or NBS1 expression levels by western blotting. One of two experiments with similar results is shown. ( b ) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (CEACAM1-KD 025), with CEACAM1 shRNA vector no. 26 (CEACAM1-KD 026) or with a control shRNA vector (CTRL) were analyzed for CEACAM1 mRNA levels by quantitative real-time PCR. Error bars indicate s.d.; n =2. ( c ) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (MCF10A CEACAM1−KD 025 ) or with a control shRNA vector (MCF10A CTRL ) were incubated in the presence or the absence of NCS 1.47 n M for the indicated time points, labeled with BrdU (10 μ M , 20 min), fixed with ice-cold ethanol and treated for analysis of the cell cycle phase distribution by flow cytometry using a FACSCalibur apparatus (Becton-Dickinson Biosciences, Le Pont-De-Claix Cedex, France). At least 10 4 events were recorded and data analysis was done with CellQuest Pro software (Becton-Dickinson Biosciences). ( d ) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (CEACAM1-KD 025), with CEACAM1 shRNA vector no. 26 (CEACAM1-KD 026) or with a control shRNA vector (CTRL) were seeded in 60 mm Petri dishes at the density of 1500 cells/dish. On the following day the cells were treated with the indicated dose of etoposide for 1 h, washed with phosphate-buffered saline, and allowed to grow in new medium at 37 °C. After 7 days, the colonies were fixed with methanol, stained with crystal violet, and counted.

Article Snippet: MCF-10A and HCT116 stable transfectants with stable CEACAM1 silencing were established using shRNA constructs against CEACAM1 in pRS vector purchased from OriGene Technologies/Labforce, Nunningen, Switzerland (cat. no. TR314008), according to the manufacturer's instructions.

Techniques: Stable Transfection, Transfection, shRNA, Plasmid Preparation, Control, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Incubation, Labeling, Flow Cytometry, Software, Saline, Staining

Journal: Oncogenesis

Article Title: The CEACAM1 tumor suppressor is an ATM and p53-regulated gene required for the induction of cellular senescence by DNA damage

doi: 10.1038/oncsis.2012.7

Figure Lengend Snippet: CEACAM1 is required for the establishment of cellular senescence as assessed by cellular morphology and senescence-associated β-galactosidase staining. ( a ) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (025) or with a control shRNA vector (CTRL) were seeded in 60 mm Petri dishes at the density of 16 000 cells/dish. On the following day the cells were treated with NCS 1.47 n M . After 6 days, the cells were photographed under phase contrast. Bar =100 μm. ( b ) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (CEACAM1-KD 025), with CEACAM1 shRNA vector no. 26 (CEACAM1-KD 026) or with a control shRNA vector (CTRL) were seeded in 6-well plates at the density of 50 000 cells/well in triplicates. On the following day the cells were treated with the indicated dose of etoposide for 1 h, washed with phosphate-buffered saline, and allowed to grow in new medium at 37 °C. After 4 days, the cells were stained for senescence-associated (SA) β-galactosidase. The graph represents the fraction of SA β-galactosidase positive cells ±s.e.m. in 15 randomly selected photographic fields where a total of at least 250 cells from two different experiments/condition were counted. p CEACAM1-KD 025 + etoposide vs CTRL + etoposide <0.001; p CEACAM1-KD 026 + etoposide vs CTRL + etoposide =0.005. Two-sided t -test. ( c ) Examples of the (SA) β-galactosidase staining of MCF-10A CTRL , MCF-10A CEACAM1−KD 025 , or MCF-10A CEACAM1−KD 026 cells treated with Etoposide as detailed and quantified in b . Bar =100 μm.

Article Snippet: MCF-10A and HCT116 stable transfectants with stable CEACAM1 silencing were established using shRNA constructs against CEACAM1 in pRS vector purchased from OriGene Technologies/Labforce, Nunningen, Switzerland (cat. no. TR314008), according to the manufacturer's instructions.

Techniques: Staining, Stable Transfection, Transfection, shRNA, Plasmid Preparation, Control, Saline

Journal: Oncogenesis

Article Title: The CEACAM1 tumor suppressor is an ATM and p53-regulated gene required for the induction of cellular senescence by DNA damage

doi: 10.1038/oncsis.2012.7

Figure Lengend Snippet: MCF-10A and HCT116 cells with stable silencing of CEACAM1 exhibit normal regulation of p21/pRb in response to DNA damage. ( a ) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (CEACAM1-KD 025) or with a control shRNA vector (CTRL) were incubated in the presence or the absence of 1.47 n M NCS for the indicated time points, lysed, and analyzed for p21/Waf1, pRb Ser 807/811-p, pRb, or β-actin by western blotting. ( b ) MCF-10A cells stably transfected with CEACAM1 shRNA vector no. 25 (CEACAM1-KD 025) or with a control shRNA vector (CTRL) were incubated in the presence or the absence of NCS 1.47 n M for 24 h in duplicate as indicated, lysed, and analyzed for p21/Waf1, pSer15-p, p53, or β-actin by western blotting. ( c ) HCT116 cells stably transfected with CEACAM1 shRNA vector no. 26 (CEACAM1-KD 026), with CEACAM1 shRNA vector no. 28 (CEACAM1-KD 028) or with a control shRNA vector (CTRL) were incubated in the presence of 20 μ M etoposide or dimethyl sulfoxide (DMSO (solvent)) for 1 h, washed with phosphate-buffered saline and incubated in new medium for 3 days. At the end of the incubation the cells were lysed and analyzed for p21/Waf1, pRb Ser 807/811-p or pRb by western blotting.

Article Snippet: MCF-10A and HCT116 stable transfectants with stable CEACAM1 silencing were established using shRNA constructs against CEACAM1 in pRS vector purchased from OriGene Technologies/Labforce, Nunningen, Switzerland (cat. no. TR314008), according to the manufacturer's instructions.

Techniques: Stable Transfection, Transfection, shRNA, Plasmid Preparation, Control, Incubation, Western Blot, Solvent, Saline