Journal: bioRxiv

Article Title: Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics

doi: 10.1101/2024.12.13.628358

Figure Lengend Snippet: (A) CD9 knockout disrupts both meningococcal and staphylococcal adherence. WT and CD9 -/- cells were infected with either meningococci (MC58) or staphylococci (SH1000) for 60 mins at an MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by colony forming units (cfu). (B-C) CD9-derived peptide, 800C, reduces meningococcal adherence (B) and staphylcoccal adherence (C) to epithelial cells. WT or CD9 -/- cells were pre-treated with a scrambled peptide or 800C for 60 mins prior to infection. Cells were infected as above and adherence enumerated by cfu. (D) Cell surface expression of meningococcal receptors measured by flow cytometry. WT or CD9 -/- cells were treated with an anti-CD9 antibody (602.29), an anti-CD46 antibody, an anti-CD147 antibody. A mouse IgG isotype control (JC1) was included and expression was determined using a FITC-conjugated secondary antibody. % change was calculated by comparing WT to CD9 -/- cells. (E) Representative blot demonstrating expression of meningococcal and staphylococcal receptors. Whole cell lysates of WT and CD9 -/- cells were electrophoresed and blotted. Blots were probed with an anti-CD44 antibody. An anti-GAPDH antibody was used as a loading control. Densitometry was calculated through ImageJ analysis, removing background with an empty lane and normalising to the loading control. n > 3, mean + SEM.

Article Snippet: Mouse anti-human CD9 IgG1 (MM2/57; Merck, Germany), mouse anti-human CD9 IgG1 (602.29; kind gift of Lynda Partridge, University of Sheffield, Sheffield, UK), mouse anti-human CD147 IgG1 (HIM6; Biolegend, California, USA), mouse anti-human CD46 IgG1 (MEM-258; Thermo Fisher Scientific), mouse anti-human CEACAM1 IgG1 (B3-17; Merck), mouse anti-human CEACAM6 IgG1 (1H7-4B; Merck), mouse anti-human CD44 IgG2a (60224-1-Ig; Proteintech Group, Inc, Illinois, USA), mouse anti-human GAPDH (MAB374; Merck), mouse IgG1 (JC1; in house), mouse IgG2a (02-6200; Thermofisher Scientific), mouse anti-FLAG (M2; Merck), goat anti-mouse HRP (P0447; Agilent, California, USA) were used as described.

Techniques: Knock-Out, Infection, Bacteria, Derivative Assay, Expressing, Flow Cytometry, Control

Journal: bioRxiv

Article Title: Dynamics of the CD9 interactome during bacterial infection of epithelial cells by proximity labelling proteomics

doi: 10.1101/2024.12.13.628358

Figure Lengend Snippet: WT cells were treated with specific siRNAs 72 hours prior to infection. Cells were treated with 800C or a scrambled control peptide for 60 minutes prior to infection. Cells were infected with either meningococci (MC58) or staphylococci (SH1000) for 60 mins at an MOI=50. (A) Flow cytometry demonstrating efficient knockdown of meningococcal and staphylococcal receptors after siRNA treatment prior to infection. Cells were probed with anti-CD147, anti-CD46 and anti-CD44 antibodies. n=1, mean. (B) CD147 knockdown and CD9-derived peptide treatment demonstrate no additive effects on meningococcal adherence. WT and CD9 -/- siRNA treated cells were infected with meningococci (MC58) for 60 mins at MOI=50. (C) CD44 knockdown and CD9-derived peptide treatment demonstrate reduced additive effects in staphylococcal adherence. WT and CD9 -/- siRNA treated cells were infected with staphylococci (SH1000) for 60 mins at MOI=50. Cells were disrupted after infection and adherent and internalised bacteria were enumerated by cfu. n=3, mean + SEM, One-Way ANOVA.

Article Snippet: Mouse anti-human CD9 IgG1 (MM2/57; Merck, Germany), mouse anti-human CD9 IgG1 (602.29; kind gift of Lynda Partridge, University of Sheffield, Sheffield, UK), mouse anti-human CD147 IgG1 (HIM6; Biolegend, California, USA), mouse anti-human CD46 IgG1 (MEM-258; Thermo Fisher Scientific), mouse anti-human CEACAM1 IgG1 (B3-17; Merck), mouse anti-human CEACAM6 IgG1 (1H7-4B; Merck), mouse anti-human CD44 IgG2a (60224-1-Ig; Proteintech Group, Inc, Illinois, USA), mouse anti-human GAPDH (MAB374; Merck), mouse IgG1 (JC1; in house), mouse IgG2a (02-6200; Thermofisher Scientific), mouse anti-FLAG (M2; Merck), goat anti-mouse HRP (P0447; Agilent, California, USA) were used as described.

Techniques: Infection, Control, Flow Cytometry, Knockdown, Derivative Assay, Bacteria

Journal: Heliyon

Article Title: Proteolytic shedding of CD46 from human hepatocytes indicates liver stress

doi: 10.1016/j.heliyon.2024.e40841

Figure Lengend Snippet: Establishing and validating an ELISA for soluble CD46 (sCD46) . (A) Signal:noise (S:N) values for different concentrations of CD46 capture and detection antibodies in Reagent Diluent 2 (n = 2). (B) Titration curve using recombinant human CD46 (rhCD46) as the analyte. Lower limit of quantification (LLOQ), upper limit of quantification (ULOQ) and limit of detection (LOD) are indicated (n = 3). (C) Accuracy (spike recovery) of human serum samples (n = 5). (D) Intra-assay precision, inter-assay precision and reproducibility of human serum sample at two different dilution factors. (E) Analysis of assay linearity (n = 4, Pearson correlation, indicating 90 % prediction bands). (F) Linear regression of the bias plotted against nominal titres (n = 4, Pearson correlation). (G) Robustness based on incubation timing (n = 4, one sample t -test to a mean of 1). (H) Stability of sCD46 (pg/ml) in representative cell culture supernatants over multiple freeze/thaw cycles depending on the storage temperature and the application of a protease inhibitor. (I) rhCD46 titration in ELISA and competitive FACS-based assay (n = 3). (J) Performance of commercial CD46 ELISA kits in detecting serum sCD46 compared to our in-house ELISA. Samples dilutions were based on manufacturers' recommendations.

Article Snippet: Next, 100 μl/well of samples, the corresponding 1:2 serial diluted standards of rhCD46 (2 ng/ml to 31.25 pg/ml, 10256-CD, R&D Systems) and a blank control are added and then incubated for 2 h. Then, 100 μl/well detection antibody (50 ng/ml, BAF2005, R&D Systems) is added followed by another incubation for 2 h. Next, 100 μl/well of Streptavidin-HRP (DY998, R&D Systems) is added followed by 100 μl/well of substrates (DY999, R&D Systems) for 20 and 30 min, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Recombinant, Intra Assay, Inter Assay, Incubation, Cell Culture, Protease Inhibitor

Journal: Heliyon

Article Title: Proteolytic shedding of CD46 from human hepatocytes indicates liver stress

doi: 10.1016/j.heliyon.2024.e40841

Figure Lengend Snippet: Establishing and validating an ELISA for soluble CD46 (sCD46) . (A) Signal:noise (S:N) values for different concentrations of CD46 capture and detection antibodies in Reagent Diluent 2 (n = 2). (B) Titration curve using recombinant human CD46 (rhCD46) as the analyte. Lower limit of quantification (LLOQ), upper limit of quantification (ULOQ) and limit of detection (LOD) are indicated (n = 3). (C) Accuracy (spike recovery) of human serum samples (n = 5). (D) Intra-assay precision, inter-assay precision and reproducibility of human serum sample at two different dilution factors. (E) Analysis of assay linearity (n = 4, Pearson correlation, indicating 90 % prediction bands). (F) Linear regression of the bias plotted against nominal titres (n = 4, Pearson correlation). (G) Robustness based on incubation timing (n = 4, one sample t -test to a mean of 1). (H) Stability of sCD46 (pg/ml) in representative cell culture supernatants over multiple freeze/thaw cycles depending on the storage temperature and the application of a protease inhibitor. (I) rhCD46 titration in ELISA and competitive FACS-based assay (n = 3). (J) Performance of commercial CD46 ELISA kits in detecting serum sCD46 compared to our in-house ELISA. Samples dilutions were based on manufacturers' recommendations.

Article Snippet: Briefly, 3 × 10 5 CD46-expressing MOLT-4 cells were stained with a PE-conjugated CD46 antibody (REA312, Miltenyi Biotec) after sequential incubation with ViaKr-808 (Beckman Coulter, USA) and 10 % human FcR block (Miltenyi Biotec).

Techniques: Enzyme-linked Immunosorbent Assay, Titration, Recombinant, Intra Assay, Inter Assay, Incubation, Cell Culture, Protease Inhibitor

Journal: Heliyon

Article Title: Proteolytic shedding of CD46 from human hepatocytes indicates liver stress

doi: 10.1016/j.heliyon.2024.e40841

Figure Lengend Snippet: Fat-induced shedding of CD46 involves the prostaglandin E2 (PGE2) pathway and matrix metalloproteinases (MMPs) . (A) Concentration of serum sCD46 in healthy (n = 112) and steatotic (n = 46) patients (Welch two sample t -test). (B) Fat-loading (FL) of HepaRG increased the concentration of sCD46 in the culture supernatants compared to unloaded (UL) HepaRG (n = 7, paired two-sample t -test). (C) The effect of MMP inhibitor TAPI-1 on CD46 shedding measured via ELISA (n = 3, Pearson correlation). (D) qPCR results expressed as fold-change in mRNA expression levels of FL/UL HepaRG (n = 6, one-sample Wilcoxon signed-rank test). (E) PGE2 treatment induces the shedding of CD46 in FL HepaRG in a dose-dependent manner (n = 3). (F) qPCR results expressed as fold-change in gene expression after PGE2 treatment (1 mM) compared to control DMSO (n = 6, Wilcoxon signed-rank test). (G) MMP1 ELISA results of FL HepaRG treated with 1 mM PGE2 or control supernatants (n = 7, Wilcoxon signedrank test).

Article Snippet: Briefly, 3 × 10 5 CD46-expressing MOLT-4 cells were stained with a PE-conjugated CD46 antibody (REA312, Miltenyi Biotec) after sequential incubation with ViaKr-808 (Beckman Coulter, USA) and 10 % human FcR block (Miltenyi Biotec).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Gene Expression, Control