Fig. 6: Intervention targeting GSDMD-mediated pyroptosis inhibits responses in keratinocytes exposed to stimulation mimicking psoriatic immune microenvironment.
From: Gasdermin D-mediated keratinocyte pyroptosis as a key step in psoriasis pathogenesis
A GSDMD siRNA or nonsense siRNA was transfected by electroporation into primary human epidermal keratinocytes. The efficiency of knockdown was measured by western blotting assay. siRNA 342 was chosen in the experiments afterwards. B Proteins of interest in cell lysate and culture supernatant were detected by western blotting assay (n = 3). C PI positive cells (red) and Ki-67 positive cells (green) were determined by immunofluorescence assay (n = 4). D–F Keratinocytes were treated by M5 for 24 h in presence or absence of DSF (40 μM). D Proteins of interest in cell lysate and culture supernatant were detected by western blotting assay (n = 3). E PI positive cells (red) and Ki-67 positive cells (green) were determined by immunofluorescence assay (n = 9). F The secretion of IL-1β, CXCL-2, CCL-20, IL-8 and S100A8/A9 were determined by ELISA (n = 3). G Keratinocytes were treated by M5 for 24 h in presence or absence of Cu(DTC)2. Proteins of interest in cell lysate and culture supernatant were detected by western blotting assay (n = 3). H, I Imiquimod-induced psoriasis-like dermatitis mice were topically applicated by 1%, 2%, 5% DSF or vehicle. H Skin appearances were presented. Histological features were analyzed by H&E staining. Immunohistochemistry study showed the levels of Ki-67. n = 3. I The cleavages of caspase-1, GSDMD and IL-1β in epidermis lysate were detected by western blotting assay (n = 3). NC: nonsense. Scale bar represents 100 μm. Scale bar represents 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001, DSF disulfiram, GSDMD-FL GSDMD full length, N-GSDMD N-terminal GSDMD, siNC nonsense control siRNA, siGSDMD GSDMD siRNA.
