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. 2024 Sep 14;15:8044. doi: 10.1038/s41467-024-51747-2

Fig. 5. HDAC3 inhibition prevents the PV-IgG-induced phenotype in vitro.

Fig. 5

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 HDAC3 inhibitors 20 μM RGFP966 (n = 4, PV-IgG DMSO vs PV-IgG RGFP966 p = 0.0002) or 20 μM Entinostat (n = 3, PV-IgG DMSO vs PV-IgG Entinostat p = 0.0004). d Immunofluorescence staining of HaCaT cells treated with Ctrl-IgG or PV-IgG and 5 μM RGFP966 or 10 μM Entinostat using DSG3, DSP antibodies and DAPI. Scale bar = 10 μm. Quantification of DSG3 intensity/membrane length of 3 independent experiments are shown. Each data point represents one cell. PV-IgG DMSO vs Ctrl-IgG DMSO p = 0.0001, PV-IgG DMSO vs PV-IgG RGFP966 p < 0.0001, PV-IgG DMSO vs PV-IgG Entinostat p < 0.0002. Values are expressed as mean with standard deviation (mean + /-SD). One n represents one biological replicate. Source data are provided as a Source Data file. All experiments were statistically analyzed with One-way-ANOVA, SIDAK correction. p < 0.05*; p < 0.01**; p < 0.001***.