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. 2023 Aug 29;220(10):e20230105. doi: 10.1084/jem.20230105

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© 2023 Robinson et al.

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Figure S1. — Additional data on the effect of EEF2 targeting bacterial toxins, including DT. (A) Anti-puromycin immunoblot of primary keratinocytes subjected to the harringtonine run-off assay. Cells were treated with BHI media, sterile filtrates of C. diphtheriae, C. striatum for 6 h, or purified DT (150 ng/ml) for 3 h before harringtonine addition (2 µg/ml). Nascent peptides were then labeled with 10 µg/ml puromycin for 10 min at different intervals. (B) Immunoblot of inflammasome components NLRP1, GSDMD, and IL-1β in N/TERT with and without TNFα priming (25 ng/ml, 8 h). Streptolysin O (SLO, 1 µg/ml) was included as additional negative control. (C) IL-1β ELISA of culture media from N/TERT cells treated with the indicated recombinant proteins or compounds. LFn (150 ng/ml): Lethal Factor from B. anthracis (aa 34–288). PA (300 ng/ml). MxiH: Shigella flexneri type 3 secretion needle protein. All cells were primed with TNFα. Significance values were calculated from one-way ANOVA with multiple group comparisons. Error bars derived from data from three technical replicates. The graph represents one of two biological replicates. (D) Kinetics of PI uptake in unprimed N/TERT cells treated with DDB/Aplidine. Significance values were calculated from Student’s t test at 4 h. Error bars are derived from data from three technical replicates. Data represent one of two biological replicates. (E) The percentage of cells with ASC-GFP specks among 293T-ASC-GFP-NLRP1 cells transfected with the indicated plasmids. Cells were fixed 24 h after transfection. ASC-GFP specks were visualized using GFP epifluorescence and normalized to the total number of cells per field of view using DAPI nuclear counterstain. Error bars are derived from data from three technical replicates. Data represent one of two biological replicates. (F) Immunoblot of overexpressed 3xFLAG-sidI and R453P glycolysase-defective mutant. Note that the level of wild-type sidI is much lower than the R453P mutant due to its strong inhibitory effect on translation, as reported previously by Subramanian et al. (2022) Preprint. (G) Immunoblot of inflammasome activator markers GSDMD and IL-1β in primary keratinocytes treated with the indicated bacterial filtrate. C. diphtheriae was cultured in BHI broth and PGT media (see Materials and methods). ***, P ≤ 0.001; ****, P ≤ 0.0001. Source data are available for this figure: SourceData FS1.