Journal: Nature Communications

Article Title: A drug-free cardiovascular stent functionalized with tailored collagen supports in-situ healing of vascular tissues

doi: 10.1038/s41467-024-44902-2

Figure Lengend Snippet: a Representative rhodamine-conjugated phalloidin (TRITC-Phalloidin, red) and 4′,6-diamidino-2-phenylindole (DAPI, blue) fluorescence images of HUVECs showing the (rhCol III/PDA-PEI) n coatings loaded with different amounts of rhCol III all encouraged HUVECs compared with control PLA group. Scale bars, 200 μm. Quantification of ( b , c ) cell number and ( d , e ) cell viability of HUVECs cultured on uncoated and (rhCol III/PDA-PEI) n -coated PLA ( n = 1, 2, and 4) sheets after 1, 3, 5, and 7 days of culture ( n = 5 independent samples). f Volcano plot showing differentially expressed genes in the (rhCol III/PDA-PEI) 2 group compared to the PLA control group. Downregulated and upregulated genes are colored blue and red, respectively, at significantly differentially expressed thresholds │log 2 FC│ > 1.2 and p < 0.05. g Gene Ontology (GO) analysis of differentially expressed genes in PLA versus (rhCol III/PDA-PEI) 2 . h Heatmap of the differentially expressed genes in the PLA and (rhCol III/PDA-PEI) 2 groups. i Circular visualization of the results of gene-annotation enrichment analysis. j Quantification of the expression of representative genes in PLA versus (rhCol III/PDA-PEI) 2 , validated by qRT-PCR arrays. The value in the PLA group was normalized to that in the (rhCol III/PDA-PEI) 2 group ( n = 3 independent samples). k Representative immunofluorescence staining of CCL5 (green), GATA3 (green), XBP1 (green), and CEACAM6 (green) of HUVECs on 3 days of culture. Scale bar, 50 µm. l Schematic diagram of the potential three signaling pathways involved in the regulation of HUVEC behavior induced by the (rhCol III/PDA-PEI) 2 coating, including PI3K/AKT, mTOR, and MAPK. Two-way ANOVA with Tukey’s multiple comparisons was used for the comparisons in ( b )–( e ) and ( j ). Two-sided Student’s t test with multiple testing corrections was used in ( f ) and ( g ). The data are presented as the mean ± SD ( p values < 0.05 were considered statistically significant).

Article Snippet: The primary antibodies used in this study included mouse monoclonal XBP1 (Cat. No.: sc-8015, Clone: F-4, Santa Cruz Biotechnology, USA, 1:50), mouse polyclonal CCL5 (Cat. No.: sc-365826, Clone: A-4, Santa Cruz Biotechnology, USA, 1:50), mouse monoclonal CEACAM6 (Cat. No.: sc-59899, Clone: 9A6, Santa Cruz Biotechnology, USA, 1:50), rabbit monoclonal GATA3 (Cat. No.: ab199428, Clone: EPR16651, Abcam, USA, 1:500), rabbit polyclonal F4/80 (Cat. No.: 29414-1-AP, Proteintech, China, 1:100), rabbit monoclonal CD68 (Cat. No.: ab283654, Clone: EPR23917-164, Abcam, USA, 1:100), rabbit polyclonal CD86 (Cat. No.: bs-1035R, Biosynthesis Biotechnology co., ltd, USA, 1:200), rabbit monoclonal CD206 (Cat. No.: 24595, Clone: E6T5J, Cell Signaling Technology, USA, 1:200), mouse monoclonal α-SMA (Cat. No.: ab7817, Clone: 1A4, Abcam, USA, 1:200), and rabbit monoclonal MMP2 (Cat. No.: 10373-2-AP, Clone: SB13a, Proteintech, USA, 1:200), mouse monoclonal CD31 (Cat. No.: ab9498, Clone: JC/70A, Abcam, USA, 1:200), and rabbit polyclonal eNOS (Cat. No.: ab5589, Abcam, USA, 1:100).

Techniques: Fluorescence, Control, Cell Culture, Expressing, Quantitative RT-PCR, Immunofluorescence, Staining, Protein-Protein interactions

Journal: Frontiers in Immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: Downregulation of IRF4 enhances CAR T cell functionality. (A) CAR T cells (starting with 1 × 10 5 CAR T cells) underwent four rounds (R1-R4) of stimulation with GFP-labeled CEA + BxPC-3 cells (1 × 10 5 tumor cells at the beginning of each round). At the end of each round, CAR T cells (live CD3 + CAR + ) (left panel) and BxPC-3 cells (right panel) were quantified by flow cytometry. Data represent means ± SEM of six donors, p values were calculated by Student´s t test, ns indicates not significant, and * indicates p ≤ 0.05. (B-G) Phenotypic analysis of CD8 + CAR T cells during repetitive antigen stimulation. CAR T cells underwent three rounds (R1-R3) of antigen-stimulation with unlabeled BxPC-3 cells. At the end of each round, the CD8/CD4 T cell ratio was determined (B) . CAR T cells were stained for CD8 and further characterized regarding TIM-3 (C) , PD-1 (D) , TIGIT (E) expression and effector-memory cell differentiation: SCM = T stem-cell-memory (CD45RO + CD62L + ), EM = effector-memory (CD45RO + CD62L - ), CM = central-memory (CD45RO + CD62L + ), E = effector (CD45RO - CD62L - ) (F) , and CD27 expression (G) . Data represent geometric means of ± SEM of at least four donors, p values were calculated by paired t test, ns indicates not significant, *indicates p ≤ 0.05, **indicates p ≤ 0.01.

Article Snippet: The density of CEA on the surface of 293T cells, BxPC3-cells and MIA PaCa-2 cells was determined using the QuantiBRITE PE assay (LOT: #60550, BD) according to the manufacturer ́s instructions in conjunction with a PE-labeled anti-CEA antibody (clone TET2, Miltenyi) using flow cytometry.

Techniques: Labeling, Flow Cytometry, Staining, Expressing, Cell Differentiation

Journal: Frontiers in Immunology

Article Title: IRF4 downregulation improves sensitivity and endurance of CAR T cell functional capacities

doi: 10.3389/fimmu.2023.1185618

Figure Lengend Snippet: Downregulation of IRF4 in CAR T cells enables killing of targets with low antigen density. (A) Staining of target cells MIA PaCa-2 for CEA expression using an APC Vio 770-conjugated anti-CEA antibody. One representative staining out of three experiments is shown. (B) T cells (1 × 10 5 T cells) with and without IRF4 downregulation were co-culture with GFP-labeled CEA + MIA PaCa-2 cells (1 × 10 5 tumor cells). After three days, CEA + MIA PaCa-2 cells (live GFP + cells) and CAR T cells (live CD3 + CAR + ) (C) were counted by flow cytometry using counting beads. Data represent means ± SEM of five donors, p values were calculated by Student’s t test, ns: not significant, **p ≤ 0.01. (D) Cytotoxicity of CAR T cells upon a 72-hour co-culture with MIA PaCa-2 cells or CEA - 293T cells was measured at the indicated effector to target cell ratios by an XTT-based colorimetric assay. Data represent means ± SEM of four donors, p values were calculated by Student´s t test. (E) IFN-γ and IL-2 in the supernatants after a 48-hour co-culture with medium (w/o), 293T cells, and MIA PaCa-2 cells was recorded by ELISA. Data represent means ± SEM of three donors, p values were calculated by Student´s t test, ns: not significant. (F) Staining of CAR T cells for CD25 and for pSTAT5 (G) after three days of co-culture with MIA-PaCa-2 cells. Data represent geometric means of ± SEM of three donors, p values were calculated by paired t test, ns indicates not significant, *indicates p ≤ 0.05. (H) ELISA-based quantification of CAR-activation induced IFN-γ and IL-2 in the supernatant after a 48-hour culture on 96 well. plates coated with the anti-idiotypic monoclonal antibody BW2064/36 at the indicated concentrations. Data represent means ± SEM of eight donors, p values were calculated by paired t test, *p ≤ 0.05.

Article Snippet: The density of CEA on the surface of 293T cells, BxPC3-cells and MIA PaCa-2 cells was determined using the QuantiBRITE PE assay (LOT: #60550, BD) according to the manufacturer ́s instructions in conjunction with a PE-labeled anti-CEA antibody (clone TET2, Miltenyi) using flow cytometry.

Techniques: Staining, Expressing, Co-Culture Assay, Labeling, Flow Cytometry, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: a , Different human cell lines were assessed for their permissivity to alphacoronavirus pseudotyped spike proteins. CcCoV-KY43 could enter Calu3 (lung) and Caco2 (intestinal) cells. The mean entry (fold change (FC) compared with non-enveloped (NE) controls) from technical triplicates is shown. b , Recombinantly produced CcCoV-KY43 RBD shows potentially high levels of glycosylation. c , Screening an array of human receptor ectodomains identified CEACAM6, CEACAM3 and CEACAM5 as interactors of the CcCoV-KY43 RBD. d , CEACAM proteins were overexpressed in refractory HEK293T cells, and the assays showed that pseudotyped CcCoV-KY43 spike protein could only enter in the presence of CEACAM6. Two biological replicates (technical triplicates) along with their s.d. values, are shown. *** P = 0.0003 using two-way analysis of variance (ANOVA). NS, not significant. e , Interaction of the CcCoV-KY43 RBD and CEACAM6 was confirmed by ELISA, whereas no binding was observed with CEACAM3 or CEACAM5. The mean of two experiments (technical triplicate) is shown with s.d. values. f , ITC (showing differential power (DP) values) of CEACAM6 and CEACAM5 binding the CcCoV-KY3 RBD. Data are representative of two (CEACAM5) or three (CEACAM6) independent experiments. For CEACAM6, mean K d , number of binding sites ( n ) and enthalpy change (Δ H ) are shown. g , Pseudotyped CcCoV-KY43 spike protein was incubated for 1 h with recombinant CEACAM6, followed by titration on HEK293T cells expressing human CEACAM6 (hCEACAM6). Raw data of the mean of two independent experiments (technical triplicate) are shown with s.d. values. h , Monoclonal antibodies against CEACAM6 (B6.2 and clone 439424) were used to neutralize CcCoV-KY43 entry into HEK293T cells expressing hCEACAM6. The mean of two independent experiments (technical triplicate) was normalized to the untreated condition and plotted with s.d. values. i , Left, CEACAM6-specific siRNAs, or negative control, were electroporated into permissive cells and then infected with CcCoV-KY43. To confirm CEACAM6 reduction, cell lysates were analysed by immunoblotting (bottom). Mean entry reduction relative to the scrambled siRNA control from three independent experiments (technical triplicates) along with s.d. values, is shown. Significance of log 10 fold change was determined using a one-sample t -test and P values were adjusted for multiple comparisons (Caco2: P = 0.004 for siRNA-1, P = 0.001 for siRNA-2 and P = 0.009 for siRNA-3; Calu3: P = 0.022 for siRNA-1, P = 0.04 for siRNA-2 and siRNA-3). Right, using lentivirus expressing shRNA, stable knockdown of CEACAM6 expression in Caco2 and Calu3 cell lines was induced and validated by immunoblotting. One biological replicate (technical triplicates) is shown, along with s.d. values. WT, wild type. j , Left, lung-specific single-cell transcriptomic data show the expression of CEACAM6. Right, comparison of the expression of coronavirus receptors in lung cells: CEACAM6, ACE2, APN, DDP4 and TMPRSS2 are shown with the dot plot indicating both average and per cent expression.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques: Produced, Glycoproteomics, Enzyme-linked Immunosorbent Assay, Binding Assay, Incubation, Recombinant, Titration, Expressing, Bioprocessing, Negative Control, Infection, Western Blot, Control, shRNA, Knockdown, Single Cell, Comparison

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: Pseudovirus (PV) entry assays showed that only CcCoV-KY43 requires CEACAM6 for entry. Experiment was performed in technical triplicate, with the mean and SD relative light units (RLU) plotted.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques:

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: HEK293T cells transfected with plasmids encoding the indicated human CEACAMs were stained using the anti-CEACAM6 commercial monoclonal antibodies, B6.2 and 493424. FITC-conjugated anti-mouse was used as secondary antibody to detect binding of the monoclonals. Surface expression was then quantified using a flow cytometer.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques: Transfection, Staining, Bioprocessing, Binding Assay, Expressing, Flow Cytometry

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: a , Structure of the CcCoV-KY43 RBD interacting with the human receptor CEACAM6. The three loops protruding from the RBD that are responsible for the interaction with CEACAM6 are highlighted. b , Open-book representation of the interfaces of interaction between the two proteins. The surface formed by residues constituting the three loops of the RBD are coloured as in a . c , Zoom-in to key residues involved in binding between the CcCoV-KY43 RBD and human CEACAM6. Residues of loop 1 (R517 and S519) and loop 2 (T552 and S555) establish hydrogen bonds with the surface polar regions of CEACAM6, whereas W600 and I605 on loop 3 of the RBD interact with the hydrophobic pocket of the receptor. d , Alignment of human CEACAM5 and CEACAM6, showing the residues interacting with CcCOV-KY43 RBD (in bold). An I63F substitution was introduced in CEACAM6, and the complementary substitution F63I in CEACAM5, followed by entry assays. The average of three independent experiments, performed in technical triplicates, is shown with s.d. values. *** P = 0.0001, **** P < 0.0001 statistical analysis using two-way ANOVA. e , Structure of alphacoronavirus RBDs in complex with human receptors used for entry: CcCoV-KY43 with CEACAM6, HCoV-229E with APN (Protein Data Bank (PDB) ID: 6ATK ) and HCoV-NL63 with ACE2 (PDB ID: 3KBH ) . Despite binding architecturally different proteins, the RBDs share similar overall folds and all bind their receptors using the same three surface loops.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques: Binding Assay

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: ( a ) Entry assays using CcCoV-KY43 S pseudotypes with HEK293T cells transiently transfected with human CEACAM5 and CEACAM6, or mutated forms of these proteins. The substitutions I63F and Q123H confirms that position 63 is a major determinant of CEACAM usage for KY43. ( b ) Flow cytometry data shows comparable expression of human CEACAM5 and CEACAM6, and their derived mutants, in HEK293T cells.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques: Transfection, Flow Cytometry, Expressing, Derivative Assay

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: Ni-NTA sensors were loaded with His-tagged CcCoV|KY43 RBD ( a ) or CcCoV-2B ( b ) and then incubated with different human CEACAMs in a two-fold concentration gradient from 600 nM (dark colours) to 37.5 nM (light colours). BLI responses following subtraction of a reference sensor incubated with buffer are shown. The end of the association phase is marked (dashed vertical line). Human CEACAM6 binds strongly to CcCoV RBDs, and this binding is lost upon introduction of an I63F substitution. CcCoV RBDs bind weakly to human CEACAM5 and displays negligible binding to CEACAM1, CEACAM3 and CEACAM8. Data are representative of two independent experiments.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques: Incubation, Concentration Assay, Binding Assay

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: a , Sites of bat sampling in Kenya and the distribution of C. cor (highlighted in teal). C. cor is found in mines (squares), natural (circles) and tree (triangles) caves and in two instances in human habitations (houses). Other sampling locations where C. cor was not observed are labelled with a cross. Inset, human population-dense areas of Kenya do not generally overlap with C. cor roosting sites. The individual sites where CcCoV were identified are shown in pale brown (CcCoV-KY43) and dark brown (CcCoV-2A and CcCoV-2B). b , Human sera from individuals ( n = 368) from Tana River and Taita-Taveta counties (highlighted in pale red in the inset map; teal indicates distribution of C. cor ) were analysed for their reactivity to different human coronavirus glycoproteins. Individual Spearman’s rank correlations ( ρ ) for each dataset (compared with CcCoV-KY43) are provided. c , Alignment of C. cor -derived alphacoronavirus RBD amino acid sequences, showing high identity in the loops interacting with CEACAM6 (loops are highlighted as in Fig. ). The secondary structure of the RBD, based on the CcCoV-KY43 RBD, is depicted above the sequences. Residues that interact with CEACAM6 are in bold. d , Entry assays with a pseudotyped spike protein of CcCoV-2A showed that human CEACAM6 confers permissivity to HEK293T cells at levels similar to CcCoV-KY43. Average of the raw data of three independent experiments, performed in technical triplicate, are shown, with s.d. values. ** P = 0.0036 for CcCoV-KY43; ** P = 0.0014 for CcCoV-2A. Statistical analysis used two-way ANOVA. e , Recombinant CcCoV-2B spike protein RBD was purified and used to assess binding to human CEACAM3, CEACAM5 and CEACAM6 by ELISA. Raw data of three independent experiments, performed in technical triplicate, are shown with s.d. values. f , ITC showed that the CcCoV-2B RBD binds CEACAM6 with high affinity. g , Crystal structure of CcCoV-2B in complex with human CEACAM6. Inset shows the CcCoV-2B and CcCoV-KY43 RBDs from the two complexes superposed, which highlights the similar folds of the RBDs and conserved interaction with the receptor. h , Zoom-in of key residues at the CEACAM6-binding interfaces of CcCoV-2B and CcCoV-KY43, highlighting the similarity of the interactions.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques: Sampling, Derivative Assay, Recombinant, Purification, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: ( a ) Pseudovirus entry of CcCoV-2A S in HEK293T cells expressing the indicated CEACAM protein. The F63I substitution in CEACAM5 increases CcCoV-2A S-mediated entry to the level seen with CEACAM6. Values are presented as relative light units (RLU). Assays were performed twice in technical triplicates with mean RLU and SD shown. ( b ) CcCoV-2A S entry into cells expressing CEACAM6 substitution I63F is reduced relative to wild type human CEACAM6 (hCEACAM6). Assays were performed twice in technical triplicates in triplicate with percentage PV entry and SD shown. Statistical analysis was performed using paired one-way ANOVA (**** p -value < 0.0001) and paired t-test (*** p -value = 0.0002), respectively.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques: Expressing

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: a , Evolutionary reconstruction of the spike protein-encoding gene for selected alphacoronaviruses demonstrates the relatively recent acquisition of CEACAM6 usage. Viruses reported to utilize human receptors are underlined. Inset map shows locations of isolation of indicated CcCoV-related viruses. b , Pseudotype entry assays for CcCoV and related viruses with CEACAM6-like proteins from different mammalian species (percentage amino acid identity compared with human CEACAM6 or CcCoV-KY43 for the full-length spike protein (FL-S) or the IgV RBD domain (RBD), respectively, is shown). Viruses closely related to CcCoV-2A, CcCoV-2B and CcCoV-KY43 users were included in the analysis, which informed the evolutionary acquisition of receptor usage. Positive results were reproduced three times, using independent biological replicates. All experiments were performed in technical triplicate with PV entry indicated as the fold change above background.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques: Isolation

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: ELISA using the recombinant RBD of btCoV-977 to assess binding to soluble Egyptian fruit bat CEACAM6-like, or human CEACAM6, proteins. The mean OD from a representative experiment, performed in technical quadruplicates, is shown together with the SD.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay

Journal: Nature

Article Title: Heart-nosed bat alphacoronaviruses use human CEACAM6 to enter cells

doi: 10.1038/s41586-026-10394-x

Figure Lengend Snippet: ( a ) Alignment of RBD sequences from the CEACAM6-using alphaCoV RBDs included in this study. Loops that interact with human CEACAM6 are highlighted in red. Residues that interact with human CEACAM6 in the CcCoV-KY43 and CcCoV-2B complex structure are shown in bold red . Residues of btCoV-LN20 that were mutated to probe the sequence determinants of human CEACAM6 usage (see below) are underlined in grey . ( b ) Host range of btCoV-LN20, a CEACAM6 user, cannot be defined by point substitutions in its RBD. Entry assays using pseudotyped btCoV-LN20 S show that single mutations in the RBD, making it more like CcCoV-KY43 S, cannot recapitulate the broad host range of CcCoV-KY43, including usage of human CEACAM6. The background-corrected average of three independent experiments, each performed in triplicate, is shown.

Article Snippet: Western blot membranes were incubated with mouse anti-human CEACAM6 (Fisher Scientific, clone B6.2, 1:1,000) and rabbit anti-human GAPDH (Proteintech, 1:10,000) as a loading control.

Techniques: Sequencing

Journal: Virulence

Article Title: Norepinephrine affects the interaction of adherent-invasive Escherichia coli with intestinal epithelial cells

doi: 10.1080/21505594.2021.1882780

Figure Lengend Snippet: Fluorescence intensity of APC-labeled anti-hCEACM6 antibody in untreated and NE-treated Caco-2 cells infected with AIEC strains (A). Star (*) indicates the statistical difference between untreated Caco-2 cells uninfected and infected with AIEC, (d) symbol indicates the statistical difference (p<0.05) between NE-treated uninfected and infected with AIEC epithelial cells, and (f) symbol indicates statistical difference (p<0.05) between untreated and NE-treated Caco-2 cells infected with AIEC (A). The expression of CEACAM6 gene in untreated and NE-treated Caco-2 cells infected with AIEC strains (B). P values were determined by Student’s two-tail t-test, *, p≤0.05. Data are the means ± SD of three independent experiments in triplicate ± SD

Article Snippet: Then, cells were stained with mouse anti-human CEACAM6/CD66 allophycocyanin (APC)-conjugated antibody (R&D, FAB3934A).

Techniques: Fluorescence, Labeling, Infection, Expressing

Journal: Heliyon

Article Title: Efficacy of bivalent CEACAM6/4- 1BBL genetic vaccine combined with anti-PD1 antibody in MC38 tumor model of mice

doi: 10.1016/j.heliyon.2022.e10775

Figure Lengend Snippet: Identification and expression of CEACAM6/4- 1BBL double genes in COS7 cells. (A) The result of reverse-transcription PCR for identification of CEACAM6/4-1BBL double genes expression in COS7 cells 48 h after transfection. Lane 1, COS7 cells transfected with pIRES-4-1BBL; 2, COS7 cells transfected with pIRES-CEACAM6; 3–4, COS7 cells transfected with pIRES-CEACAM6/4-1BBL; 5, COS7 cells transfected with pIRES vector plasmids; 6, COS7 cells transfected with nothing (negative); 7–8, positive control of pIRES-CEACAM6/4-1BBL plasmids. 4- 1BBL: 930 bp; forward primer: 5-GCTCTAGAGCCACCATGGACCAGCACGCACTTG-3; reverse primer: 5-GGCGGCCGCGTCATCCCTGAGGGGGGTC-3. CEACAM6 : 801 bp; forward primer: 5-CAGAGCCAAACAACAGAT-3; reverse primer: 5-CATTATTACTTATGCTGACCT-3. Primary uncropped image of (A) is shown in supplementary Figure S1. (B) Representative Western blots showing the successful expression of CEACAM6/4-1BBL proteins in COS7 cell line 48 h after transfection. Control: COS7 cells transfected with vector plasmids. Double-gene plasmid: COS7 cells transfected with pIRES-CEACAM6/4-1BBL plasmids. (C) Western blotting images showing the expression of 4-1BBL and CEACAM6 proteins in the intestine and tumor tissues of mice treated or not treated with the bivalent genetic vaccine of pIRES-CEACAM6-4-1BBL/SL3261. GAPDH was used as an internal reference. Primary uncropped images of (B) and (C) are shown in supplementary Figure S2.

Article Snippet: Antibodies used: anti-4-1BBL mouse monoclonal antibody (sc-398933, Santa cruz, 1:1000), anti-CEACAM6 rabbit monoclonal antibody (ab235139, Abcam, 1:1000), anti-PD-L1 rabbit polyclonal antibody (ab233482, Abcam, 1:1000), and anti-GAPDH mouse monoclonal antibody (ab125247, Abcam, 1:5000), and secondary antibodies (Beyotime Biotechnology).

Techniques: Expressing, Transfection, Plasmid Preparation, Positive Control, Western Blot

Journal: Heliyon

Article Title: Efficacy of bivalent CEACAM6/4- 1BBL genetic vaccine combined with anti-PD1 antibody in MC38 tumor model of mice

doi: 10.1016/j.heliyon.2022.e10775

Figure Lengend Snippet: Drug efficacy of anti-PD1 antibody combined with CEACAM6/4- 1BBL genetic vaccine. (A) Statistical result of the therapeutical effect of CEACAM6/4-1BBL vaccine, and PD1 antibody (Nivolumab) combined with vaccine, on the growth of MC38 heterotopic tumor in mice (averages of tumor volume ratios, and SD). Treatments started at 2 weeks after heterotopic tumor transplantation. Control, given vehicle. Vaccine, given the bivalent vaccine of pIRES-CEACAM6-4-1BBL/SL3261 alone. Combined, treated with bivalent vaccine and PD1 antibody (Nivolumab, 20 mg/kg) together. (B) Representative images of isolated MC38 tumors of the control (2490 mm 3 ), vaccine (2147), and combined immunotherapy (1181) groups. N = 4 per time point. ∗∗∗P < 0.001, Combined vs. Control; #P < 0.05, Combined vs. Vaccine.

Article Snippet: Antibodies used: anti-4-1BBL mouse monoclonal antibody (sc-398933, Santa cruz, 1:1000), anti-CEACAM6 rabbit monoclonal antibody (ab235139, Abcam, 1:1000), anti-PD-L1 rabbit polyclonal antibody (ab233482, Abcam, 1:1000), and anti-GAPDH mouse monoclonal antibody (ab125247, Abcam, 1:5000), and secondary antibodies (Beyotime Biotechnology).

Techniques: Transplantation Assay, Isolation

Journal: Heliyon

Article Title: Efficacy of bivalent CEACAM6/4- 1BBL genetic vaccine combined with anti-PD1 antibody in MC38 tumor model of mice

doi: 10.1016/j.heliyon.2022.e10775

Figure Lengend Snippet: Tumor tissue PD1/PD-L1 expression after treatment with CEACAM6/4- 1BBL vaccine and anti-PD1 immunotherapy. (A) Representative immunohistochemical images showing the expression of PD-1 and PD-L1 in MC38 tumor tissue after treatment. Control, given vehicle; Vaccine alone, given the bivalent vaccine; Combined immunotherapy, given bivalent vaccine and anti-PD1 drug (Nivolumab). Tissue samples were collected at 2 weeks after treatments. Purple, hematoxylin staining for nucleus; brown, anti-PD1 or anti-PD-L1 staining. Scale bars, 100 μm. (B) Statistical results of the mean integral optical density (MIOD, IOD/Area) of the IHC images. N = 3 per group. ∗P < 0.05, ∗∗∗P < 0.001 vs. Control; #P < 0.05, ###P < 0.001 vs. Vaccine alone.

Article Snippet: Antibodies used: anti-4-1BBL mouse monoclonal antibody (sc-398933, Santa cruz, 1:1000), anti-CEACAM6 rabbit monoclonal antibody (ab235139, Abcam, 1:1000), anti-PD-L1 rabbit polyclonal antibody (ab233482, Abcam, 1:1000), and anti-GAPDH mouse monoclonal antibody (ab125247, Abcam, 1:5000), and secondary antibodies (Beyotime Biotechnology).

Techniques: Expressing, Immunohistochemical staining, Staining