nrcam  (Alomone Labs)

Journal: eBioMedicine

Article Title: Reduced circulating NrCAM as a biomarker for fetal growth restriction

doi: 10.1016/j.ebiom.2025.105854

Figure Lengend Snippet: Bar graphs and receiver operator curves of Neuronal Cell Adhesion Molecule (NrCAM) in two large cohorts ( a-d , Cohort 1, e-h , Cohort 2). NrCAM concentrations significantly reduced ( a , p = 4.75 x 10 −6 ) in the circulation of women at 36 weeks' gestation who subsequently delivered an FGR infant (birthweight <3rd centile). The discriminatory power of NrCAM is shown as a receiver operating characteristic (AUROC) curve with an area of the curve of 0.76 ( b ). Plasma Placental Growth Factor ( c, d ; PlGF) concentrations at 36 weeks were also significantly reduced in the same cohort ( c , p = 8.15 × 10 −7 ). The AUROC of PlGF is 0.77 ( d ). Cohort, n = 957 controls, n = 26 FGR. In a high-risk international cohort (FEMINA, Manchester, UK) ( e , f ) show NrCAM concentrations reduced ( e , p = 9.34 x 10 −3 ) in the circulation of women who presented at the clinic with reduced fetal movements who subsequently delivered an FGR infant (birthweight <3rd centile), AUROC of 0.72 ( f ). Plasma PlGF ( g, h ) concentrations in the same cohort remain unchanged ( g , p = 0.13). The AUROC of PlGF is 0.63 ( h ). Cohort, n = 235 controls, n = 12 FGR. groups compared using Mann–Whitney U tests. AUROC, area under the AUROC curve, with 95% confidence intervals presented in brackets. Data depicted for a , c, e and g are mean ± SEM. ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Article Snippet: Taqman Fast Advanced Master Mix (Applied Biosystems) and specific fluorescein amidite (FAM)- labelled Taqman Gene Expression Assays (Life Technologies) were used to measure the gene expression of human NRCAM (Neuronal Cell Adhesion Molecule, Assay ID: Hs01031598_m1), NFASC (Neurofascin, Assay ID: Hs00391791_m1), TEAD4 (TEA Domain Transcription Factor 4, Assay ID: Hs01125032_m1), CDH2 (Cadherin-2, Assay ID: Hs00983056_m1), SDC1 (Syndecan 1, Assay ID: Hs00896423_m1) and HLAG (Human Leucocyte Antigen G, Assay ID: Hs03045108_m1).

Techniques: Clinical Proteomics, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Reduced circulating NrCAM as a biomarker for fetal growth restriction

doi: 10.1016/j.ebiom.2025.105854

Figure Lengend Snippet: Circulating Neuronal Cell Adhesion Molecule (NrCAM) ( a , b ) concentrations were not altered ( a , p = 0.09) in the circulation of 24 women at 36 weeks' gestation who subsequently developed preeclampsia (PE) at term, compared with 936 controls. The discriminatory power of NrCAM is shown as a receiver operating characteristic curve with a modest area of the curve (AUROC) of 0.60 ( b ). Plasma Placental Growth Factor ( c, d ; PlGF) concentration is significantly reduced in participants who develop preeclampsia at term ( c , p = 5.45 x 10 −8 ). The AUROC of PlGF is 0.84 ( d ). Controls, n = 959 controls, n = 24 PE. In samples from a case control cohort (PROVE, South Africa), circulating NrCAM was significantly reduced ( e , p = 0.03) in participants with severe preeclampsia. The AUROC of 0.70 ( f ). NrCAM is not altered in participants with eclampsia ( g ), with a modest AUROC of 0.59 ( h ). Control, n = 15 controls, n = 27 preeclampsia (PE), n = 29 eclampsia. Groups compared using Mann–Whitney U tests. AUROC, area under the ROC curve, with 95% confidence intervals presented in brackets. Data depicted for a , c, e and f are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Article Snippet: Taqman Fast Advanced Master Mix (Applied Biosystems) and specific fluorescein amidite (FAM)- labelled Taqman Gene Expression Assays (Life Technologies) were used to measure the gene expression of human NRCAM (Neuronal Cell Adhesion Molecule, Assay ID: Hs01031598_m1), NFASC (Neurofascin, Assay ID: Hs00391791_m1), TEAD4 (TEA Domain Transcription Factor 4, Assay ID: Hs01125032_m1), CDH2 (Cadherin-2, Assay ID: Hs00983056_m1), SDC1 (Syndecan 1, Assay ID: Hs00896423_m1) and HLAG (Human Leucocyte Antigen G, Assay ID: Hs03045108_m1).

Techniques: Clinical Proteomics, Concentration Assay, Control, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Reduced circulating NrCAM as a biomarker for fetal growth restriction

doi: 10.1016/j.ebiom.2025.105854

Figure Lengend Snippet: Circulating Neuronal Cell Adhesion Molecule (NrCAM) concentrations were significantly reduced in a case control of participants with preterm fetal growth restriction (FGR, <34 weeks' gestation), compared to gestation-matched controls ( a , p = 0.0003). Gestation-matched controls, n = 20, FGR, n = 23. The discriminatory power of NrCAM is shown as a receiver operating characteristic (AUROC) of 0.82 ( c ). Circulating NrCAM is reduced in participants with preterm preeclampsia (<34 weeks' gestation), compared to gestation-matched controls ( b , p = 0.0003). Gestation-matched controls, n = 20, preeclampsia, n = 41. The AUROC is 0.79 ( d ). NrCAM protein concentrations are reduced in placenta lysates from participants who delivered an FGR infant ( e , p = 0.005). Gestation-matched controls, n = 19, FGR, n = 43. NrCAM protein concentrations are reduced in placenta lysates from participants who are diagnosed with preeclampsia ( f, p = 0.0002), compared to gestation-matched controls. Gestation-matched controls, n = 21, preeclampsia, n = 27. NRCAM mRNA expression is not altered in placenta from participants who delivered an FGR infant ( g ) or diagnosed with preeclampsia ( h ) compared with preterm controls. Gestation-matched controls, n = 17, FGR, n = 63, preeclampsia, n = 78. NFASC mRNA expression is significantly reduced in the placenta from participants who delivered an FGR infant ( i, p = 0.03) or developed preeclampsia at term ( j , p = 0.003), compared with gestation-matched controls. Gestation-matched controls, n = 18, FGR, n = 30, preeclampsia, n = 78. Groups compared using Mann–Whitney U tests. Data presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Taqman Fast Advanced Master Mix (Applied Biosystems) and specific fluorescein amidite (FAM)- labelled Taqman Gene Expression Assays (Life Technologies) were used to measure the gene expression of human NRCAM (Neuronal Cell Adhesion Molecule, Assay ID: Hs01031598_m1), NFASC (Neurofascin, Assay ID: Hs00391791_m1), TEAD4 (TEA Domain Transcription Factor 4, Assay ID: Hs01125032_m1), CDH2 (Cadherin-2, Assay ID: Hs00983056_m1), SDC1 (Syndecan 1, Assay ID: Hs00896423_m1) and HLAG (Human Leucocyte Antigen G, Assay ID: Hs03045108_m1).

Techniques: Control, Expressing, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Reduced circulating NrCAM as a biomarker for fetal growth restriction

doi: 10.1016/j.ebiom.2025.105854

Figure Lengend Snippet: NRCAM mRNA expression is reduced following differentiation of cytotrophoblast stem cells to syncytiotrophoblast and extravillous trophoblast (EVT) cells. hTSCs were differentiated into syncytiotrophoblast and EVT cells from 0 h to 96 h. To confirm EVT differentiation, reduction in cytotrophoblast marker TEAD4 ( a , p = 0.006 72 h, p = 0.001 96 h), and increase in EVT marker HLAG ( b , p = 0.02 72 h, p = 0.0002 96 h) mRNA expression was observed. To confirm syncytiotrophoblast differentiation, reduction in cytotrophoblast marker TEAD4 ( d ), and increase in syncytiotrophoblast marker SDC1 ( e ) mRNA expression was observed. NRCAM mRNA expression was reduced in EVT ( c ) and syncytiotrophoblast ( f ) following differentiation. NRCAM mRNA expression was reduced when cytotrophoblast hTSC cells ( g ), but not syncytiotrophoblast cells ( h ) exposed to hypoxia (1% O 2 ), compared to control physiological conditions (normoxia; 8% O 2 ). NRCAM mRNA expression was reduced in primary trophoblast cells ( i ) exposed to hypoxia (1% O 2 ) compared to normoxia (8% O 2 ). mRNA expression was normalized to the geometric mean of housekeeper genes. All experiments were repeated 5 times in triplicate. In primary term human trophoblast cells, NRCAM mRNA expression was significantly reduced in cells exposed to hypoxia (1% O 2 ), compared to normoxic (8% O 2 ) controls ( e , normoxia, n = 6, hypoxia n = 6). For data with two groups, unpaired t-test or a Mann–Whitney (non-parametric) test was used. For 3 or more groups, a one-way ANOVA (parametric) or a Kruskal Wallis (nonparametric) test was used. For tests using multiple comparison tests, data was compared to the control group. Data was presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Taqman Fast Advanced Master Mix (Applied Biosystems) and specific fluorescein amidite (FAM)- labelled Taqman Gene Expression Assays (Life Technologies) were used to measure the gene expression of human NRCAM (Neuronal Cell Adhesion Molecule, Assay ID: Hs01031598_m1), NFASC (Neurofascin, Assay ID: Hs00391791_m1), TEAD4 (TEA Domain Transcription Factor 4, Assay ID: Hs01125032_m1), CDH2 (Cadherin-2, Assay ID: Hs00983056_m1), SDC1 (Syndecan 1, Assay ID: Hs00896423_m1) and HLAG (Human Leucocyte Antigen G, Assay ID: Hs03045108_m1).

Techniques: Expressing, Marker, Control, MANN-WHITNEY, Comparison

Journal: eBioMedicine

Article Title: Reduced circulating NrCAM as a biomarker for fetal growth restriction

doi: 10.1016/j.ebiom.2025.105854

Figure Lengend Snippet: In a mouse model of hypoxia-induced fetal growth restriction, fetal weight ( a ) is significantly reduced in hypoxia treated mothers (10% inspired O 2 ), compared to normoxia controls (21% inspired O 2 ). Placental weight remained unchanged ( b ), whilst placenta-to-body ratio is significantly increased in maternal hypoxia treated group, compared with normoxic controls ( c ). NrCAM mRNA expression is significantly decreased in placentas from the maternal hypoxia group, compared with normoxic controls ( d , n = 9 normoxic placentas, n = 9 hypoxic placentas from separate litters, n = 9 mice in each group). Data was presented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Taqman Fast Advanced Master Mix (Applied Biosystems) and specific fluorescein amidite (FAM)- labelled Taqman Gene Expression Assays (Life Technologies) were used to measure the gene expression of human NRCAM (Neuronal Cell Adhesion Molecule, Assay ID: Hs01031598_m1), NFASC (Neurofascin, Assay ID: Hs00391791_m1), TEAD4 (TEA Domain Transcription Factor 4, Assay ID: Hs01125032_m1), CDH2 (Cadherin-2, Assay ID: Hs00983056_m1), SDC1 (Syndecan 1, Assay ID: Hs00896423_m1) and HLAG (Human Leucocyte Antigen G, Assay ID: Hs03045108_m1).

Techniques: Expressing

Journal: eBioMedicine

Article Title: Reduced circulating NrCAM as a biomarker for fetal growth restriction

doi: 10.1016/j.ebiom.2025.105854

Figure Lengend Snippet: Bar graphs and receiver operator curves of Neuronal Cell Adhesion Molecule (NrCAM) in two large cohorts ( a-d , Cohort 1, e-h , Cohort 2). NrCAM concentrations significantly reduced ( a , p = 4.75 x 10 −6 ) in the circulation of women at 36 weeks' gestation who subsequently delivered an FGR infant (birthweight <3rd centile). The discriminatory power of NrCAM is shown as a receiver operating characteristic (AUROC) curve with an area of the curve of 0.76 ( b ). Plasma Placental Growth Factor ( c, d ; PlGF) concentrations at 36 weeks were also significantly reduced in the same cohort ( c , p = 8.15 × 10 −7 ). The AUROC of PlGF is 0.77 ( d ). Cohort, n = 957 controls, n = 26 FGR. In a high-risk international cohort (FEMINA, Manchester, UK) ( e , f ) show NrCAM concentrations reduced ( e , p = 9.34 x 10 −3 ) in the circulation of women who presented at the clinic with reduced fetal movements who subsequently delivered an FGR infant (birthweight <3rd centile), AUROC of 0.72 ( f ). Plasma PlGF ( g, h ) concentrations in the same cohort remain unchanged ( g , p = 0.13). The AUROC of PlGF is 0.63 ( h ). Cohort, n = 235 controls, n = 12 FGR. groups compared using Mann–Whitney U tests. AUROC, area under the AUROC curve, with 95% confidence intervals presented in brackets. Data depicted for a , c, e and g are mean ± SEM. ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Article Snippet: Taqman fast advanced Master Mix (Applied Biosystems) and FAM-labelled Taqman Gene expression Assays (Applies Biosystems) to measure the gene expression of mouse NRCAM (Neuronal Cell Adhesion Molecule, Assay ID: Mm00663607_m1). qRT-PCR was performed on the CFX384 (Bio-Rad) with thermocycling parameters: 95 °C for 20 s, 40 cycles of denaturation for 3 s at 95 °C and 60 °C for 30 s. No product was detected in the non-template control and gene expression of human samples and the hTSCs were normalised to the geometric mean of CYC1 (Cytochrome C1, Assay ID: Hs00357717_m1) and TOP1 (DNA Topoisomerase I, Assay ID: Hs00243257_m1) housekeepers.

Techniques: Clinical Proteomics, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Reduced circulating NrCAM as a biomarker for fetal growth restriction

doi: 10.1016/j.ebiom.2025.105854

Figure Lengend Snippet: Circulating Neuronal Cell Adhesion Molecule (NrCAM) ( a , b ) concentrations were not altered ( a , p = 0.09) in the circulation of 24 women at 36 weeks' gestation who subsequently developed preeclampsia (PE) at term, compared with 936 controls. The discriminatory power of NrCAM is shown as a receiver operating characteristic curve with a modest area of the curve (AUROC) of 0.60 ( b ). Plasma Placental Growth Factor ( c, d ; PlGF) concentration is significantly reduced in participants who develop preeclampsia at term ( c , p = 5.45 x 10 −8 ). The AUROC of PlGF is 0.84 ( d ). Controls, n = 959 controls, n = 24 PE. In samples from a case control cohort (PROVE, South Africa), circulating NrCAM was significantly reduced ( e , p = 0.03) in participants with severe preeclampsia. The AUROC of 0.70 ( f ). NrCAM is not altered in participants with eclampsia ( g ), with a modest AUROC of 0.59 ( h ). Control, n = 15 controls, n = 27 preeclampsia (PE), n = 29 eclampsia. Groups compared using Mann–Whitney U tests. AUROC, area under the ROC curve, with 95% confidence intervals presented in brackets. Data depicted for a , c, e and f are mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

Article Snippet: Taqman fast advanced Master Mix (Applied Biosystems) and FAM-labelled Taqman Gene expression Assays (Applies Biosystems) to measure the gene expression of mouse NRCAM (Neuronal Cell Adhesion Molecule, Assay ID: Mm00663607_m1). qRT-PCR was performed on the CFX384 (Bio-Rad) with thermocycling parameters: 95 °C for 20 s, 40 cycles of denaturation for 3 s at 95 °C and 60 °C for 30 s. No product was detected in the non-template control and gene expression of human samples and the hTSCs were normalised to the geometric mean of CYC1 (Cytochrome C1, Assay ID: Hs00357717_m1) and TOP1 (DNA Topoisomerase I, Assay ID: Hs00243257_m1) housekeepers.

Techniques: Clinical Proteomics, Concentration Assay, Control, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Reduced circulating NrCAM as a biomarker for fetal growth restriction

doi: 10.1016/j.ebiom.2025.105854

Figure Lengend Snippet: Circulating Neuronal Cell Adhesion Molecule (NrCAM) concentrations were significantly reduced in a case control of participants with preterm fetal growth restriction (FGR, <34 weeks' gestation), compared to gestation-matched controls ( a , p = 0.0003). Gestation-matched controls, n = 20, FGR, n = 23. The discriminatory power of NrCAM is shown as a receiver operating characteristic (AUROC) of 0.82 ( c ). Circulating NrCAM is reduced in participants with preterm preeclampsia (<34 weeks' gestation), compared to gestation-matched controls ( b , p = 0.0003). Gestation-matched controls, n = 20, preeclampsia, n = 41. The AUROC is 0.79 ( d ). NrCAM protein concentrations are reduced in placenta lysates from participants who delivered an FGR infant ( e , p = 0.005). Gestation-matched controls, n = 19, FGR, n = 43. NrCAM protein concentrations are reduced in placenta lysates from participants who are diagnosed with preeclampsia ( f, p = 0.0002), compared to gestation-matched controls. Gestation-matched controls, n = 21, preeclampsia, n = 27. NRCAM mRNA expression is not altered in placenta from participants who delivered an FGR infant ( g ) or diagnosed with preeclampsia ( h ) compared with preterm controls. Gestation-matched controls, n = 17, FGR, n = 63, preeclampsia, n = 78. NFASC mRNA expression is significantly reduced in the placenta from participants who delivered an FGR infant ( i, p = 0.03) or developed preeclampsia at term ( j , p = 0.003), compared with gestation-matched controls. Gestation-matched controls, n = 18, FGR, n = 30, preeclampsia, n = 78. Groups compared using Mann–Whitney U tests. Data presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Taqman fast advanced Master Mix (Applied Biosystems) and FAM-labelled Taqman Gene expression Assays (Applies Biosystems) to measure the gene expression of mouse NRCAM (Neuronal Cell Adhesion Molecule, Assay ID: Mm00663607_m1). qRT-PCR was performed on the CFX384 (Bio-Rad) with thermocycling parameters: 95 °C for 20 s, 40 cycles of denaturation for 3 s at 95 °C and 60 °C for 30 s. No product was detected in the non-template control and gene expression of human samples and the hTSCs were normalised to the geometric mean of CYC1 (Cytochrome C1, Assay ID: Hs00357717_m1) and TOP1 (DNA Topoisomerase I, Assay ID: Hs00243257_m1) housekeepers.

Techniques: Control, Expressing, MANN-WHITNEY

Journal: eBioMedicine

Article Title: Reduced circulating NrCAM as a biomarker for fetal growth restriction

doi: 10.1016/j.ebiom.2025.105854

Figure Lengend Snippet: NRCAM mRNA expression is reduced following differentiation of cytotrophoblast stem cells to syncytiotrophoblast and extravillous trophoblast (EVT) cells. hTSCs were differentiated into syncytiotrophoblast and EVT cells from 0 h to 96 h. To confirm EVT differentiation, reduction in cytotrophoblast marker TEAD4 ( a , p = 0.006 72 h, p = 0.001 96 h), and increase in EVT marker HLAG ( b , p = 0.02 72 h, p = 0.0002 96 h) mRNA expression was observed. To confirm syncytiotrophoblast differentiation, reduction in cytotrophoblast marker TEAD4 ( d ), and increase in syncytiotrophoblast marker SDC1 ( e ) mRNA expression was observed. NRCAM mRNA expression was reduced in EVT ( c ) and syncytiotrophoblast ( f ) following differentiation. NRCAM mRNA expression was reduced when cytotrophoblast hTSC cells ( g ), but not syncytiotrophoblast cells ( h ) exposed to hypoxia (1% O 2 ), compared to control physiological conditions (normoxia; 8% O 2 ). NRCAM mRNA expression was reduced in primary trophoblast cells ( i ) exposed to hypoxia (1% O 2 ) compared to normoxia (8% O 2 ). mRNA expression was normalized to the geometric mean of housekeeper genes. All experiments were repeated 5 times in triplicate. In primary term human trophoblast cells, NRCAM mRNA expression was significantly reduced in cells exposed to hypoxia (1% O 2 ), compared to normoxic (8% O 2 ) controls ( e , normoxia, n = 6, hypoxia n = 6). For data with two groups, unpaired t-test or a Mann–Whitney (non-parametric) test was used. For 3 or more groups, a one-way ANOVA (parametric) or a Kruskal Wallis (nonparametric) test was used. For tests using multiple comparison tests, data was compared to the control group. Data was presented as mean ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: Taqman fast advanced Master Mix (Applied Biosystems) and FAM-labelled Taqman Gene expression Assays (Applies Biosystems) to measure the gene expression of mouse NRCAM (Neuronal Cell Adhesion Molecule, Assay ID: Mm00663607_m1). qRT-PCR was performed on the CFX384 (Bio-Rad) with thermocycling parameters: 95 °C for 20 s, 40 cycles of denaturation for 3 s at 95 °C and 60 °C for 30 s. No product was detected in the non-template control and gene expression of human samples and the hTSCs were normalised to the geometric mean of CYC1 (Cytochrome C1, Assay ID: Hs00357717_m1) and TOP1 (DNA Topoisomerase I, Assay ID: Hs00243257_m1) housekeepers.

Techniques: Expressing, Marker, Control, MANN-WHITNEY, Comparison

Journal: eBioMedicine

Article Title: Reduced circulating NrCAM as a biomarker for fetal growth restriction

doi: 10.1016/j.ebiom.2025.105854

Figure Lengend Snippet: In a mouse model of hypoxia-induced fetal growth restriction, fetal weight ( a ) is significantly reduced in hypoxia treated mothers (10% inspired O 2 ), compared to normoxia controls (21% inspired O 2 ). Placental weight remained unchanged ( b ), whilst placenta-to-body ratio is significantly increased in maternal hypoxia treated group, compared with normoxic controls ( c ). NrCAM mRNA expression is significantly decreased in placentas from the maternal hypoxia group, compared with normoxic controls ( d , n = 9 normoxic placentas, n = 9 hypoxic placentas from separate litters, n = 9 mice in each group). Data was presented as mean ± SEM. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Article Snippet: Taqman fast advanced Master Mix (Applied Biosystems) and FAM-labelled Taqman Gene expression Assays (Applies Biosystems) to measure the gene expression of mouse NRCAM (Neuronal Cell Adhesion Molecule, Assay ID: Mm00663607_m1). qRT-PCR was performed on the CFX384 (Bio-Rad) with thermocycling parameters: 95 °C for 20 s, 40 cycles of denaturation for 3 s at 95 °C and 60 °C for 30 s. No product was detected in the non-template control and gene expression of human samples and the hTSCs were normalised to the geometric mean of CYC1 (Cytochrome C1, Assay ID: Hs00357717_m1) and TOP1 (DNA Topoisomerase I, Assay ID: Hs00243257_m1) housekeepers.

Techniques: Expressing

Journal: mBio

Article Title: Identification of the Staphylococcus aureus endothelial cell surface interactome by proximity labeling

doi: 10.1128/mbio.03654-24

Figure Lengend Snippet: Identification of ASM-dependent host co-receptors for S. aureus by an APEX2-based proximity labeling screen. (A) Decorated bacteria are still internalized by host cells in an ASM-dependent manner. HuLEC was treated with amitriptyline, ionomycin, and β-toxin and then infected with APEX2-decorated S. aureus JE2. Invasion efficiency was determined by lysostaphin protection assay and colony-forming units (CFU) recovery assays. Numbers of bacteria internalized by treated cells were normalized to untreated controls (set to 100%, dotted line). (B–D) Establishment of the ASM and Ca 2+ -dependent host-pathogen interactome. HuLEC was pretreated with either amitriptyline, the bacterial SMase β-toxin, and ionomycin or left untreated. Cells were infected with APEX2-decorated S. aureus , and interaction partners were determined by proximity labeling. (B) Abundance of individual proteins was compared to untreated controls, and median log2 fold changes were determined ( n = 3). (C) Proteins with a median log2 fold change ≤−1 were selected as candidate targets for each condition. (D) A list of candidate receptors that were downregulated in at least six of nine replicates (regardless of the treatment) were considered as “consistently reduced.” (E and F) Blockade of NRCAM and MFI2 with antibodies reduces the invasion efficiency of S. aureus . HuLEC was pretreated with antibodies targeting NRCAM (E) or melanotransferrin (MFI2), NT5E/CD73 (F), or the respective solvent control. Then, cells were infected with S. aureus for 10 min, and invasion efficiency was determined by a lysostaphin protection assay and CFU counting ( n ≥ 4). (G) S. aureus invasion is reduced in a PTK7 knockout (K.O.) cell line. The invasion efficiency of S. aureus JE2 after 10 min or 30 min was determined in a HeLa cell line lacking PTK7 and was compared to WT cells ( n = 7). (H) S. aureus invasion is reduced in CD109 and MET K.O. cell lines. The invasion efficiency of S. aureus JE2 after 10 min was determined in a HeLa cell line lacking CD109 ( n = 9), and MET ( n = 5) and compared to WT cells. Statistics: unpaired Student‘s t -test (E and G) or one sample t -test (F and H). * P ≤ 0.05, ** P ≤ 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: 30 ng/mL anti-NRCAM antibody , Proteintech/21608–1-AP , 75 min , Removed prior to infection.

Techniques: Labeling, Bacteria, Infection, Solvent, Control, Knock-Out