pcdna3 1 zeo expression vector  (Thermo Fisher)

Journal: Communications Biology

Article Title: Gasdermin-D pores induce an inactivating caspase-4 cleavage that limits IL-18 production in the intestinal epithelium

doi: 10.1038/s42003-025-08183-9

Figure Lengend Snippet: HCT116 epithelial cells were treated with 2 μM of pan-caspase inhibitor Z-VAD-FMK ( a – c ), siRNA targeting caspase-4 ( d ) or with caspase-1/4 specific inhibitor VX-765 ( g – i ). C2bbe1 epithelial cells were transduced with shRNA or transfected with siRNA as indicated to generate double -caspase-1 or -5 and GSDMD knockdowns ( e , f ). For inflammasome activation, cells were electroporated with 2 μg LPS. HEK293T cells ( k – m ) or CASP4 –/– HCT116 cells were transfected with caspase-4 expression vectors indicated in ( j ). HEK293T cells were harvested 16 h post transfection ( k – m , p ). HCT116 cells were electroporated with LPS 16 h post transfection ( n , o ). Cell lysis was measured by LDH cytotoxicity assay ( a , k ). Western blots were conducted on whole protein lysates ( g , l , n , p ). IL-18 release was measured by ELISA on supernatant alone ( b , h ) or by lysing cells into supernatant prior to ELISA ( c , d , e , f , i , m , o ). Representative of at least 3 experiments except for panel ( p ) ( N = 1), data displayed as mean ± S.E.M. **** p < 0.001, *** p < 0.005.

Article Snippet: pcDNA3.1 vectors expressing human caspase-4, human IL-18 and human GSDMD were purchased from Genscript.

Techniques: Transduction, shRNA, Transfection, Activation Assay, Expressing, Lysis, LDH Cytotoxicity Assay, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Communications Biology

Article Title: Gasdermin-D pores induce an inactivating caspase-4 cleavage that limits IL-18 production in the intestinal epithelium

doi: 10.1038/s42003-025-08183-9

Figure Lengend Snippet: a–g HCT116 cells were pre-treated with 50 μM dimethylfumerate (DMF) for 1 h then electroporated with LPS and placed back into media containing 50 μM DMF. h–l GSDMD and mutants indicated in schematic ( h ) were generated. GFG contains point mutations at W48 and W50, that prevent insertion of pores into the plasma membrane. L192A is cleaved and forms pores with less efficiency that WT GSDMD, D275A cannot be cleaved, L304A/L308A cannot bind the caspase-4 exosite. Mutants were transiently transfected into HEK293T cells along with WT caspase-4 and IL-18. Cell lysis was measured by LDH ( a ), GSDMD, caspase-4 or IL-18 cleavage was measured by western blots on whole cell lysates or supernatant as indicated ( b , e ). IL-18 was measured by ELISA on supernatant alone ( c ), or on cells lysed into supernatant ( e , g , k ). Cellular viability was measured by CellTitre-Glo assay® at indicated timepoints for ( f ) or 16 h following transfection for ( j ). PI uptake was measured by calculating the mean fluorescence intensity (MFI) of each condition compared to non-transfected control ( i ). Representative of at least 3 experiments, data displayed as mean ± S.E.M. **** p < 0.001*** p < 0.005, ** p < 0.01, * p < 0.05. Panel ( h ) was created with Biorender.

Article Snippet: pcDNA3.1 vectors expressing human caspase-4, human IL-18 and human GSDMD were purchased from Genscript.

Techniques: Generated, Clinical Proteomics, Membrane, Transfection, Lysis, Western Blot, Enzyme-linked Immunosorbent Assay, Glo Assay, Fluorescence, Control

Journal: Communications Biology

Article Title: Gasdermin-D pores induce an inactivating caspase-4 cleavage that limits IL-18 production in the intestinal epithelium

doi: 10.1038/s42003-025-08183-9

Figure Lengend Snippet: a – e HCT116 cells were electroporated then placed in media with 10 mM glycine to prevent cell lysis. In panels ( f – i ), HCT116 cells were silenced for NINJ1 expression using shRNA constructs, as indicated, before LPS electroporation. Cell lysis was measured by LDH cytotoxicity assay ( a , g ). GSDMD ( b ) and caspase-4 ( e , i ) cleavage was measured by western blots of whole cell lysates. IL-18 was measured by ELISA on supernatants ( c ), or on cells lysed into supernatant ( d , h ). The effect of NINJ1 shRNA constructs 1 and 5 on NINJ1 expression in HCT116 cells was determined by qRT-PCR ( f ). Green arrow indicates LPS dependent caspase-4 cleavage products. Representative of at least 3 experiments (except for ( h ) where n = 2), data displayed as mean ± S.E.M. * p < 0.05.

Article Snippet: pcDNA3.1 vectors expressing human caspase-4, human IL-18 and human GSDMD were purchased from Genscript.

Techniques: Lysis, Expressing, shRNA, Construct, Electroporation, LDH Cytotoxicity Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Communications Biology

Article Title: Gasdermin-D pores induce an inactivating caspase-4 cleavage that limits IL-18 production in the intestinal epithelium

doi: 10.1038/s42003-025-08183-9

Figure Lengend Snippet: Human duodenal organoids were trypsinised into single cells and seeded onto culture plates pre-coated with 1:40 Matrigel:PBS. Cells were infected with S. flexneri ∆ ospc3 at an MOI of 100 for 4 h. CFUs in infected cells were enumerated ( a ), PI uptake was monitored using incucyte live cell imager and PI uptake was normalised per area confluency ( b ), images of monolayers taken 4 h post infection taken at 20x (i) scramble, uninfected; (ii) scramble, Shigella flexneri ∆ ospc3, (iii) GSDMD KD uninfected, (iv) GSDMD KD Shigella flexneri ∆ ospc3. Scale bars (indicting 200 micrometres) are depicted for each micrograph ( c ). Caspase-4 was measured in western blot of whole cell lysates ( d ), IL-18 secretion into the supernatant was measured by ELISA on cell free supernatants ( e ). A schematic of caspase-4 regulation ( f ). In ( f ) (i) LPS-induced caspase-4 activation (1) leads to activation of GSDMD and IL-18 (2). GSDMD pores lead to caspase-4 cleavage (3) and this stops IL-18 processing (4). In ( f )(ii), during conditions of reduced GSDMD –mediated plasma pore formation, caspase-4 activation (1) leads to IL-18 processing. A lack of efficient pore formation reduces inhibitory feedback on caspase-4 (3). This leads to continued activity of caspase-4 on IL-18 and IL-18 hypersecretion (4). All experiments are representative of at least 3 experiments. Data displayed as mean ± S.E.M. * p < 0.05, **** p < 0.001. Panel ( f ) created with Biorender.

Article Snippet: pcDNA3.1 vectors expressing human caspase-4, human IL-18 and human GSDMD were purchased from Genscript.

Techniques: Infection, Western Blot, Enzyme-linked Immunosorbent Assay, Activation Assay, Clinical Proteomics, Activity Assay