Journal: Frontiers in Immunology

Article Title: Claudin-2 enhances human antibody-mediated complement-dependent cytotoxicity of porcine endothelial cells by modulating antibody binding and complement activation

doi: 10.3389/fimmu.2025.1547512

Figure Lengend Snippet: In PIECs, Claudin-2 did not affect the expression of CD46, CD55, CD59, Factor H, or Factor I. (A) PIECs were infected with control lentivirus (ShNC) or with lentivirus expressing Claudin-2 specific siRNA(ShCLDN2-1, or ShCLDN2-2). After 4 days, total RNA was collected and the mRNA level of CD46, CD55, CD59, Factor H, or Factor I was analyzed by RT-PCR. (B, C) PIECs were treated as in A and analyzed with flow cytometry to assess the protein level of CD46 (B) . The quantitation data were presented by geometric mean fluorescence intensity (Gmean) (C) . (D) PIECs were infected with Adv-EV, or Adv-Claudin-2 for 72h, the total RNA was collected and the mRNA level of CD46, CD55, CD59, Factor H, or Factor I was analyzed by RT-PCR. (E, F) PIECs were treated as in D and analyzed with flow cytometry to assess the protein level of CD46 (E) . The quantitation data were presented by Gmean (F) . Data are representative of at least three independent experiments (mean ± SEM). N.S. means no significance.

Article Snippet: Anti-pig CD46 antibody was from Bio-Rad (Hercules, CA, USA).

Techniques: Expressing, Infection, Control, Reverse Transcription Polymerase Chain Reaction, Flow Cytometry, Quantitation Assay, Fluorescence

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