Journal: Viruses

Article Title: Recombinant Pseudorabies Virus with TK/gE Gene Deletion and Flt3L Co-Expression Enhances the Innate and Adaptive Immune Response via Activating Dendritic Cells

doi: 10.3390/v13040691

Figure Lengend Snippet: Characterization of the recombinant HNX-TK − /gE − -Flt3L. ( A ) Detection of the transcription of the Flt3L gene in HNX-TK − /gE − -Flt3L virus- and HNX-infected cells through RT-qPCR (**** < 0.001). ( B ) Detection of the Flt3L protein and viral gB protein expression through an immunofluorescence assay (IFA). Green: gB-positive cells; red: Flt3L-positive cells; blue: DAPI-stained PK-15 cell nucleus. DAPI, 4’,6-diamidino-2-phenylindole. ( C ) Western blot analysis of β-actin, gD, and Flt3L of the PK-15 cells infected with HNX-TK − /gE − -Flt3L, HNX, or DMEM as a control. ( D ) One step growth curves of HNX-TK − /gE − -Flt3L and HNX in PK-15 cells.

Article Snippet: The blots were blocked by skim milk (BD, Bioscience, Inc., Saint Louis, MO, USA), and this was followed by incubation with rabbit anti-Flt3L polyclonal antibody (Bioss, Beijing, China), mouse anti-gD monoclonal antibody (Keqian Ltd., Wuhan, China), and mouse anti-β-actin monoclonal antibody (Bioss).

Techniques: Recombinant, Infection, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Western Blot

Journal: Scientific Reports

Article Title: New lymphatic cell formation is associated with damaged brain tissue clearance after penetrating traumatic brain injury

doi: 10.1038/s41598-021-89616-3

Figure Lengend Snippet: Immunohistochemical staining images showing CD34 expression in ( A ) normal mouse brain tissue and ( B – F ) mouse brain tissue on days 3, 7, 15, 21 and 30 days, following the pTBI. ( A ) CD34 was not expressed in normal mouse brain tissue. CD34-positive cells and blood vessels (brown; black arrows) became visible at the injury site (red arrow) on days ( B ) 3 and ( C ) 7. ( D ) After 15 days, CD34 expression at the injury site (red arrow) decreased. ( E ) CD34 expression at the injury site could not be detected on day 21. Instead, black hemosiderin particles (black arrows) were observed at the injury site. ( F ) Some black hemosiderin particles remained visible at the injury site on day 30. ( G ) Immunohistochemical results of CD34 were analyzed by Image-Pro Plus 6.0 software. The average density (IOD) of CD34-positive cells in normal mouse brain tissue and in mouse brain tissue on days 3, 7, 15, 21 and 30 days, following pTBI. The n = 10/group was used for calculating the average IOD. Comparisons between groups were performed using the Kruskal–Wallis H test followed by Bonferroni post-hoc analysis.

Article Snippet: The sections were first incubated with rabbit anti-mouse PROX1 antibody (1:100; Boster Biological Technology Co., Wuhan, China), rabbit anti-mouse CD34 antibody (1:100; BosterBiological Technology) or goat anti-mouse LYVE-1 antibody (1:100; R&D Systems, Shanghai, China) at 4 °C for 24 h. Next, the sections were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG or rabbit anti-goat IgG secondary antibodies.

Techniques: Immunohistochemical staining, Staining, Expressing, Software

Journal: Scientific Reports

Article Title: New lymphatic cell formation is associated with damaged brain tissue clearance after penetrating traumatic brain injury

doi: 10.1038/s41598-021-89616-3

Figure Lengend Snippet: Double immunofluorescence staining images showing CD34/LYVE-1 expression in the brain tissue 3 days after pTBI. Expression of ( A ) LYVE-1 (green); ( B ) CD34 (red); and ( C ) merge (yellow).

Article Snippet: The sections were first incubated with rabbit anti-mouse PROX1 antibody (1:100; Boster Biological Technology Co., Wuhan, China), rabbit anti-mouse CD34 antibody (1:100; BosterBiological Technology) or goat anti-mouse LYVE-1 antibody (1:100; R&D Systems, Shanghai, China) at 4 °C for 24 h. Next, the sections were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG or rabbit anti-goat IgG secondary antibodies.

Techniques: Double Immunofluorescence Staining, Expressing

Journal: Journal of Clinical Medicine

Article Title: A Specific Tubular ApoA-I Distribution Is Associated to FSGS Recurrence after Kidney Transplantation

doi: 10.3390/jcm10102174

Figure Lengend Snippet: Localization of ApoA-I and megalin, a specific marker of the proximal tubular brush border, in recurrent FSGS and non-FSGS kidney allograft biopsies. ApoA-I and megalin were detected in kidney allograft biopsies by immunofluorescence using specific antibodies and nuclei were counterstained with Hoechst 33342. The figure shows a representative image of the megalin (in green), ApoA-I (in red) and Hoechst (in blue) staining obtained in recurrent-FSGS and non-FSGS patients; as well as the merged image. Megalin staining (in green) localized at the brush border of the proximal tubular cells in both recurrent FSGS and non-FSGS patients. Regarding ApoA-I, relapsing FSGS patients showed a strong ApoA-I-megalin colocalization, which was not observed in non-FSGS patients (see details of each case in the merged image zoom box).

Article Snippet: The sections were then blocked with 5% bovine serum albumin for 1 h at room temperature and incubated with antibodies against ApoA-I (dilution 1:1000 of rabbit polyclonal PAB8546, Abnova, Jhongli, Taiwan) and megalin (dilution 1:50 of mouse monoclonal MAB9578, R&D Systems, Minneapolis, MN, USA) overnight at 4 °C.

Techniques: Marker, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: Angiotensin II induces skin fibrosis: a novel mouse model of dermal fibrosis

doi: 10.1186/ar4028

Figure Lengend Snippet: Primary and secondary antibodies for immunofluorescence staining

Article Snippet: Next, cells were incubated at 4°C overnight with primary antibodies: goat anti-mouse VE-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-mouse FSP1 (Abcam, Cambridge, MA, USA).

Techniques: Immunofluorescence

Journal: bioRxiv

Article Title: The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult gut

doi: 10.1101/2021.04.14.439776

Figure Lengend Snippet: a , Intestinal cells from adult wild type mice were isolated and cultured for 2 passages in Matrigel. Matrigel derived organoids were then seeded on a collagen layer for at least 2 passages before RNAseq (n = organoids from 3 different mice), scale bar = 200um. b , Volcanoplot shows significantly (padj <0.05) upregulated (blue) and downregulated (red) genes in collagen vs. Matrigel cultures. The 30 most significant deregulated genes and intestinal stem cell genes ( Lgr5, Olfm4, Lrig1 ) are labelled. Fetal-like genes and intestinal stem cell genes are highlighted in orange. c , Geneset enrichment analysis was performed with reactome pathway signatures and fetal-organoid signature . The top 5 up- and down-regulated pathways (ordered by NES) are shown. d , Fetal signature is highly enriched in collagen cultures. e , Collagen cultures were plated on different matrices, collagen with/without addition of Matrigel or pure Matrigel, scale bar = 100 um. f , Immunofluorescence of collagen+50% Matrigel shows crypt-like protrusions with apical accumulation of Actin, containing Lysozyme+ Paneth cells and were surrounded by AldolaseB+ enterocytes, scale bar = 100 um. g , qRT-PCR shows decrease of fetal-like genes ( Col4a1, Ly6a, Msln ) and upregulation of intestinal stem cell genes ( Lgr5, Olfm4 ) and Paneth cell genes ( Lyz2 ). Each dot represents an independent experiment, bar height = mean, error bar = s.e.m.

Article Snippet: TaqMan assays and TaqMan Gene Expression Master Mix (Applied Biosystems) were used to measure gene expression: LamC1 (Mm00711820_m1), Anxa1 (Mm00440225_m1), Col4a1 (Mm01210125_m1), Ly6a (Mm00726565_s1), Msln (Mm00450770_m1), Lyz1 (Mm00657323_m1), Olfm4 (Mm01320260_m1), and LGR5 (Mm00438890_m1).

Techniques: Isolation, Cell Culture, Derivative Assay, Immunofluorescence, Quantitative RT-PCR

Journal: bioRxiv

Article Title: The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult gut

doi: 10.1101/2021.04.14.439776

Figure Lengend Snippet: a , Clustering of Matrigel derived organoids at day 4 after passaging. Frequency plot shows proportion of cells assigned to each cluster. Heatmap shows top 30 differentially expressed genes in each cluster and labels of characteristic genes of intestinal cell types. b , Clustering of collagen derived cells at day 4 after passaging. Frequency plot shows proportion of cells assigned to each cluster. Heatmap shows top 30 differentially expressed genes in each cluster and labels of characteristic genes of intestinal cell types. Fetal-like genes are labelled purple. Cells for Matrigel and collagen scRNAseq were derived from the same organoid line. c , Ly6a expression in tSNE of collagen cultures. d , IF of collagen culture, stained for nuclei (Hoechst), proliferation (Ki67) and fetal-like gene (Ly6a). Scale bar = 100 um. e , Flow Cytometry of Ly6a-APC in Matrigel and collagen cultures. Each dot represents an independent experiment, bar height = mean, error bar = s.e.m., n = 5. f , Ly6a negative and positive cells from collagen cultures were sorted and plated in Matrigel. Organoids counted after 1 week, each dot represents an independent experiment, bar height = mean, error bar = s.e.m., n = 3. g , Immunofluorescence of organoids derived from Ly6a sorted cells, stained for nuclei (Hoechst - blue), Actin (red) and Paneth cells (Lysozyme - yellow). Scale bar = 100 um.

Article Snippet: TaqMan assays and TaqMan Gene Expression Master Mix (Applied Biosystems) were used to measure gene expression: LamC1 (Mm00711820_m1), Anxa1 (Mm00440225_m1), Col4a1 (Mm01210125_m1), Ly6a (Mm00726565_s1), Msln (Mm00450770_m1), Lyz1 (Mm00657323_m1), Olfm4 (Mm01320260_m1), and LGR5 (Mm00438890_m1).

Techniques: Derivative Assay, Passaging, Expressing, Staining, Flow Cytometry, Immunofluorescence

Journal: bioRxiv

Article Title: The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult gut

doi: 10.1101/2021.04.14.439776

Figure Lengend Snippet: a , Matrigel mainly consists of Laminin and Collagen IV. Single basement membrane components (laminin/entactin or collagen IV) were added to a 1mg/ml collagen hydrogel. b , Cells grown on collagen were plated on collagen I + laminin (1.5 mg/ml, 3 mg/ml, 5mg/ml) or + Matrigel (5 mg/ml), or collagen or Matrigel alone. Laminin induces morphological changes, as well as a similar downregulation of fetal-like genes ( Col4a1, Ly6a, Msln ) and upregulation of stem cell markers ( Lgr5, Olfm4 ), n = 2-4 independent samples per mixture, black scale bar = 200um, white scale bar = 50um. Black arrow indicates Paneth cells in crypt-like protrusion. c , Cells grown on collagen were plated on collagen I, collagen I + 1.5 mg/ml collagen IV, collagen I + 5 mg/ml Matrigel or Matrigel alone. n = 3. Scale bar = 100um. d , qRT-PCR analysis of intestinal tissue at different timepoints during development, N=5 mice per time point, height = mean, error bar = s.e.m. e , Heatmap of qRT-PCR averages scaled for each gene showing that all basement membrane genes cluster together, separate from collagen I ( Col1a1 ) and are specifically upregulated at P0 (birth) and P7. Intestinal stem cell genes ( Lgr5, Olfm4 ) are upregulated at P7. Paneth cell genes ( Lyz1 ) appear later at P14, and increase further until P42. f , Laminin immunofluorescence shows increased laminin levels during crypt development, n = 3 mice, scale bar = 45um. g , Intestinal crypts (without villi) at P7 was EDTA-separated for epithelium and mesenchyme. Vimentin shows enrichment of mesenchymal cells that are absent in the epithelial fraction. Laminin and collagen IV genes are highly expressed in the mesenchyme with little contribution of epithelial cells, Error bars = s.e.m. h , RNA in situ hybridization (RNAscope) for Lamc1 and Col4a1 at P0 shows predominant mesenchymal expression, scale bar = 50um. For all bar graphs (b, c, g), each dot represents an independent experiment, bar height = mean, error bar = s.e.m.

Article Snippet: TaqMan assays and TaqMan Gene Expression Master Mix (Applied Biosystems) were used to measure gene expression: LamC1 (Mm00711820_m1), Anxa1 (Mm00440225_m1), Col4a1 (Mm01210125_m1), Ly6a (Mm00726565_s1), Msln (Mm00450770_m1), Lyz1 (Mm00657323_m1), Olfm4 (Mm01320260_m1), and LGR5 (Mm00438890_m1).

Techniques: Membrane, Quantitative RT-PCR, Immunofluorescence, RNA In Situ Hybridization, RNAscope, Expressing

Journal: bioRxiv

Article Title: The extracellular matrix controls stem cell specification and crypt morphology in the developing and adult gut

doi: 10.1101/2021.04.14.439776

Figure Lengend Snippet: Parallels between in vitro cultures with different ECMs and in vivo crypt formation and homeostasis. In vitro , adult intestinal cells contain fetal-like cells (Ly6a+) and few intestinal stem cells (Lgr5+) and grow exclusively in a 2D layer. Addition of laminin induces an increase of Lgr5+ cells and Paneth cells (via ITGA6), resulting in a morphological crypt formation. In vivo , the mesenchyme dramatically remodels the ECM and increases laminin concentration. Concurringly, epithelial cells in the inter-villus region upregulate expression of the laminin receptor ITGA6 and progress to form intestinal crypts. Even in the adult, intestinal crypts rely on the laminin signalling, as depletion of laminin results in a fetal-like epithelium.

Article Snippet: TaqMan assays and TaqMan Gene Expression Master Mix (Applied Biosystems) were used to measure gene expression: LamC1 (Mm00711820_m1), Anxa1 (Mm00440225_m1), Col4a1 (Mm01210125_m1), Ly6a (Mm00726565_s1), Msln (Mm00450770_m1), Lyz1 (Mm00657323_m1), Olfm4 (Mm01320260_m1), and LGR5 (Mm00438890_m1).

Techniques: In Vitro, In Vivo, Concentration Assay, Expressing

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: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

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: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

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: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

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: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

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: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

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: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

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: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

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: To validate the screen results, top- and bottom-strand oligos of control (gNTC) or the top three CEACAM1 gRNAs were synthesized and cloned into the library backbone plasmid lentiGuide-Puro (Addgene #52963) using a Golden Gate assembly as described .

Techniques: Expressing, Activity Assay, Binding Assay

Journal: Frontiers in microbiology

Article Title: Validation of flavivirus infectious clones carrying fluorescent markers for antiviral drug screening and replication studies.

doi: 10.3389/fmicb.2023.1201640

Figure Lengend Snippet: FIGURE 1 Design of the wt and reporter containing the icDNA of flaviviruses. (A) Schematic of the viral constructs used in the study. The reporter (mCherry, oxGFP, or NLuc) and foot-and-mouth-disease virus 2A sequence (2A) were placed at the junction of two full-length capsid sequences (shown with slanted lines). The second sequence was codon-altered using synonymous substitutions to avoid homologous recombination. (B) Rescue and growth

Article Snippet: For the staining, cells were permeabilized with PBS containing 0.1% Triton X-100 (SigmaAldrich, France) for 5 min and blocked with PBS containing 2% FBS and 0.05% Tween 20 (Sigma-Aldrich, France) for 1 h. Cells were washed once with PBS and incubated with a mouse pan-flavivirus anti-Env antibody (Mab 4G2, Novus Biologicals NBP2) for 1–2 h at room temperature (1:1,000) for viral titration or an anti-GFP antibody (A11122, Invitrogen) for the measurement of oxGFP-positive cells.

Techniques: Construct, Virus, Sequencing, Homologous Recombination

Journal: Frontiers in microbiology

Article Title: Validation of flavivirus infectious clones carrying fluorescent markers for antiviral drug screening and replication studies.

doi: 10.3389/fmicb.2023.1201640

Figure Lengend Snippet: FIGURE 2 Evaluation of the genetic stability of -mCherry, -oxGFP-, and -NLuc reporter-carrying flaviviruses. (A) Schematic of the experimental protocol used to perform the marker stability assay. Vero cells were infected at an MOI 1 with P0 stocks of recombinant ZIKV, KUNV, DENV2, or DENV4 with mCherry, oxGFP, or NLuc markers and incubated until the development of CPE or visible marker expression. P1 stocks were collected and used to infect Vero cells for the next passage until passage 4 (P1 to P4 stocks). Viral stocks were titrated using an FFA with a mouse pan-flavivirus anti-Env antibody (Mab 4G2). mCherry signal was measured by quantifying fluorescence using a Cellomics ArrayScan VTI microscope. oxGFP expression was detected by an immunofluorescence assay using an anti-GFP antibody. For the measurement of NanoLuciferase activity, 96-well plate was infected with P1 to P4 stocks according to the end-point titration method, and NLuc was measured using the Nano-Glo Luciferase Assay System after cell lysis, on a microplate reader. (B) Evolution of transgene expression through serial passages for each virus. The data represent the expression of the marker reported to the infectious titer. One-way ANOVA followed by the LSD post-hoc test was used to evaluate the significance of the relationship. Each bar represents the mean ± SD (n = 3). *p < 0.05, ***p < 0.001, ****p < 0.0001. LSD, least significant difference.

Article Snippet: For the staining, cells were permeabilized with PBS containing 0.1% Triton X-100 (SigmaAldrich, France) for 5 min and blocked with PBS containing 2% FBS and 0.05% Tween 20 (Sigma-Aldrich, France) for 1 h. Cells were washed once with PBS and incubated with a mouse pan-flavivirus anti-Env antibody (Mab 4G2, Novus Biologicals NBP2) for 1–2 h at room temperature (1:1,000) for viral titration or an anti-GFP antibody (A11122, Invitrogen) for the measurement of oxGFP-positive cells.

Techniques: Marker, Stability Assay, Infection, Recombinant, Incubation, Expressing, Fluorescence, Microscopy, Immunofluorescence, Activity Assay, Titration, Luciferase, Lysis, Virus

Journal: Frontiers in microbiology

Article Title: Validation of flavivirus infectious clones carrying fluorescent markers for antiviral drug screening and replication studies.

doi: 10.3389/fmicb.2023.1201640

Figure Lengend Snippet: FIGURE 5 Antiviral activity of NITD008 on the reporter-expressing flaviviruses. (A) The dose response of NITD008 against ZIKV-mCherry was monitored by fluorescence microscopy. Each panel corresponds to a mosaic image (six tiles) of a single well within a 96-well plate with Vero cells incubated with the indicated increasing concentrations (0.05, 0.1, 0.5, 1, 2.5, 5, and 15 μM) of NITD008 and infected with ZIKV-mCherry at an MOI of 0.05. Upper line,

Article Snippet: For the staining, cells were permeabilized with PBS containing 0.1% Triton X-100 (SigmaAldrich, France) for 5 min and blocked with PBS containing 2% FBS and 0.05% Tween 20 (Sigma-Aldrich, France) for 1 h. Cells were washed once with PBS and incubated with a mouse pan-flavivirus anti-Env antibody (Mab 4G2, Novus Biologicals NBP2) for 1–2 h at room temperature (1:1,000) for viral titration or an anti-GFP antibody (A11122, Invitrogen) for the measurement of oxGFP-positive cells.

Techniques: Activity Assay, Expressing, Fluorescence, Microscopy, Incubation, Infection