Journal: Cell Death Discovery

Article Title: Disulfiram alleviates pristane-induced lupus via inhibiting GSDMD-mediated pyroptosis

doi: 10.1038/s41420-022-01167-2

Figure Lengend Snippet: A Effect of DSF on the cell viability of THP-1 cells. Cells were treated with DSF (0, 1, 5, 10, 20 μM) for 48 h and then cell viability measured by CCK-8 assay. B The morphological features of THP-1 cells treated with serums from healthy controls or SLE patients, with or without mixing 10 μM disulfiram. C Lactate dehydrogenase (LDH) release from THP-1 cells treated as indicated. D Hoechst33342/Propidium Iodide (PI) double staining in THP-1 cells after different treatments. E Representative immunofluorescence images showing the expression of NLRP3, caspase-1, and cleaved caspase-1 in THP-1 cells treated as indicated. F The expression of full length and cleaved GSDMD in THP-1 cells. The cells were incubated in different mediums and analyzed by western blot analysis. β-actin was used as a protein loading control. G , H The expression level of total GSDMD and cleaved-GSDMD relative to β-actin were quantified. Total GSDMD = full length-GSDMD + cleaved-GSDMD. Significant differences were calculated using one-way ANOVA. Values were shown as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Each experiment was repeated three times. FBS fetal bovine serum, HC Healthy control, NS not significant, PI propidium iodide, LDH Lactate dehydrogenase.

Article Snippet: Antibodies included mouse GSDMD (Affinity Biologicals, catalog AF4012, Ancaster, Canada), human GSDMD and cleaved GSDMD (Abcam, catalog ab215203 and ab210070, Cambridge, MA, USA), β-actin (ProteinTech Group, catalog 66009, Chicago, IL), horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, catalog 111-035-003 and 115-035-003, West Grove, PA), NLRP3 (Affinity Biologicals, catalog DF7438, Ancaster, Canada), caspase-1 (Santa Cruz Biotechnology, catalog sc392736, Dallas, TX, USA), cleaved caspase-1 (Affinity Biologicals, catalog AF4022, Ancaster, Canada), F4/80 (Santa Cruz Biotechnology, catalog sc377009, Dallas, TX, USA), IgG conjugated to Alexa Fluor 555 (Abcam, catalog ab150114, Cambridge, MA, USA) and rabbit anti-mouse C3 antibody (Abcam, catalog ab97462 Cambridge, MA, USA).

Techniques: CCK-8 Assay, Double Staining, Immunofluorescence, Expressing, Incubation, Western Blot

Journal: Cell Death Discovery

Article Title: Disulfiram alleviates pristane-induced lupus via inhibiting GSDMD-mediated pyroptosis

doi: 10.1038/s41420-022-01167-2

Figure Lengend Snippet: A Representative immunohistochemical staining images of NLRP3, caspase-1, cleaved caspase-1 and GSDMD in the kidneys. B Double immunofluorescence staining of glomerular macrophages (green) and GSDMD (red) in each group. F4/80 represented macrophages. C GSDMD in monocytes/macrophages as a proportion of total GSDMD was calculated. D The level of serum IL-1β in each group was detected by ELISA. n = 8 animals for each group. Each experiment was repeated three times. Significant differences were calculated using one-way ANOVA. Values were shown as mean ± SD. ** p < 0.01, *** p < 0.001. PIL pristane-induced lupus. DAPI 4′, 6-diamidino-2-phenylindole.

Article Snippet: Antibodies included mouse GSDMD (Affinity Biologicals, catalog AF4012, Ancaster, Canada), human GSDMD and cleaved GSDMD (Abcam, catalog ab215203 and ab210070, Cambridge, MA, USA), β-actin (ProteinTech Group, catalog 66009, Chicago, IL), horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, catalog 111-035-003 and 115-035-003, West Grove, PA), NLRP3 (Affinity Biologicals, catalog DF7438, Ancaster, Canada), caspase-1 (Santa Cruz Biotechnology, catalog sc392736, Dallas, TX, USA), cleaved caspase-1 (Affinity Biologicals, catalog AF4022, Ancaster, Canada), F4/80 (Santa Cruz Biotechnology, catalog sc377009, Dallas, TX, USA), IgG conjugated to Alexa Fluor 555 (Abcam, catalog ab150114, Cambridge, MA, USA) and rabbit anti-mouse C3 antibody (Abcam, catalog ab97462 Cambridge, MA, USA).

Techniques: Immunohistochemical staining, Staining, Double Immunofluorescence Staining, Enzyme-linked Immunosorbent Assay