Journal: Nature communications

Article Title: Unbiased screening identifies regulators of cell-cell adhesion and treatment options in pemphigus.

doi: 10.1038/s41467-024-51747-2

Figure Lengend Snippet: Fig. 2 | KLF5 modulates DSG3 protein levels and cell–cell adhesion. a Flow cytometry analysis of HaCaT cells stained with anti-DSG3 antibodies or IgG as control. HaCaT cells were transduced with sgNT, sgKLF5_1 (p = 0.0698), or sgKLF5_2 (p = 0.0206). A representative histogram and mean fluorescence intensity of four independent experiments are displayed. b Western blot analysis of HaCaT cell lysates using KLF5, DSG3, and GAPDH antibodies. HaCaT cells were stably trans- duced with sgNT, sgKLF5_1, or sgKLF5_2. Representative Western blot images and quantifications of the respective proteins are shown; sgKLF_1 (p = 0.0005, n = 7), sgKLF5_2 (p = 0.0023, n = 6). Values were normalized to sgNT. c Quantitative Real- time PCR analysis of DSG3 of mRNA extracted from HaCaT cells stably transduced with sgNT, sgKLF5_1, or sgKLF5_2. Values were normalized to sgNT. n = 6, sgKLF5_1 p = 0.0235; sgKLF5_2 p < 0.0001. d Immunofluorescence staining of HaCaT cells stably transduced with sgNT, sgKLF5_1 or sgKLF5_2 using DSG3 antibodies and DAPI as nuclear stain (n = 3, sgKLF5_1 p < 0.0001, sgKLF5_2 p = 0.0009). Representative

Article Snippet: On the day of sorting, 50 × 106 cells were collected as a control (termed presort), and 60 × 106 cells were stained with Alexa 647 anti-human Dsg3 antibody (Santa Cruz Biotechnology, sc-53487 AF647) or with PE antihuman CD324 antibody (BD BioLegend, #324106) for fluorescenceactivated cell sorting (FACS) with the BD InfluxTM Cell Sorter (BD Biosciences).

Techniques: Flow Cytometry, Staining, Control, Transduction, Western Blot, Stable Transfection, Real-time Polymerase Chain Reaction

Journal: Nature communications

Article Title: Unbiased screening identifies regulators of cell-cell adhesion and treatment options in pemphigus.

doi: 10.1038/s41467-024-51747-2

Figure Lengend Snippet: Fig. 5 | HDAC3 inhibition prevents the PV-IgG-induced phenotype in vitro. a Dispase-based dissociation assay of HaCaT cells treated with Ctrl or PV-IgG and indicated concentrations of HDAC3 inhibitor RGFP966. Representative images and quantifications of (n = 5, Ctrl-IgG vs PV-IgG p < 0.0001, PV-IgG vs PV-IgG 1 μM RGFP966 p < 0.0001, PV-IgG vs PV-IgG 5 μM RGFP966 p < 0.0001) are shown. b Western blot analysis of HaCaT cell lysates using DSG3 and GAPDH antibodies. HaCaT cells were treated for 24 h with Ctrl-IgG or PV-IgG and DMSO or RGFP966. Representative Western blot images and quantifications of respective proteins (Ctrl-IgG DMSO n = 8, Ctrl-IgG RGFP966 n = 4 , PV-IgG DMSO n = 8, PV-IgG RGFP966 n = 8, PV-IgG DMSO vs PV-IgG 1 µM RGFP966 p = 0.0340) are shown. Values were normalized to IgG DMSO. c Luciferase assay (luciferase activity expressed in RLU - relative luminescence units) of HaCaT cells treated with Ctrl-IgG or PV-IgG and

Article Snippet: On the day of sorting, 50 × 106 cells were collected as a control (termed presort), and 60 × 106 cells were stained with Alexa 647 anti-human Dsg3 antibody (Santa Cruz Biotechnology, sc-53487 AF647) or with PE antihuman CD324 antibody (BD BioLegend, #324106) for fluorescenceactivated cell sorting (FACS) with the BD InfluxTM Cell Sorter (BD Biosciences).

Techniques: Inhibition, In Vitro, Western Blot, Luciferase, Activity Assay