Journal: Communications Biology

Article Title: A human cell-free translation screen identifies the NT-2 mycotoxin as a ribosomal inhibitor that binds the peptidyl transferase center

doi: 10.1038/s42003-026-10743-6

Figure Lengend Snippet: A Puromycin incorporation assay in HEK293T cells treated with increasing NT-2 concentrations, DMSO, or CHX. Equal amounts of cell lysate were loaded into a 4–12% Bis-Tris gel and analysed by Western blot probing with anti-puromycin and anti-GAPDH antibodies. B Quantification of protein synthesis based on signal intensity in the anti-Puromycin-stained blot ( A ) normalized with GAPDH. The calculated IC 50 for NT-2 in living cells was 135 nM, based on the average of three biological replicates (dots) ± SD. C Polysome profiles of NT-2-treated HEK293T cells (2 h with 2 μM NT-2 or DMEM+/+ medium). D Western blot analysis of eIF2α and phospho-eIF2α in NT-2–treated cells. HEK293T cells were treated with NT-2 (2 μM) or 0.02% DMSO (CTR) for 2 h. Equal amounts of cell lysate were analysed by Western blot probing with anti-Vinculin, anti-eIF2α and anti-phospho-eIF2α antibodies. An equal amount of HeLa S3 lysate was loaded as a positive control for phosphorylation, as described in ref. . E Growth curve of E.coli treated with 8 µM NT-2 toxin, 0.08% DMSO, or tetracycline (translation-inhibition control). F Cell-free translation reactions with Renilla luciferase mRNA in E.coli S30 lysate in the presence of no supplement (CTR), 5 µM NT-2, 50 µM NT-2, cycloheximide (CHX, 100 µg/ml), tetracycline (Tet, 10 µg/ml), or without RNA. The measured luminescence was normalized to the control reaction (CTR). Data are presented as mean ± SD values of three biological replicates (sets of translation reactions) averaged after three measurements. G Growth curves of S. cerevisiae treated with 25 µM NT-2, 0.25% DMSO, or 10 µg/ml CHX as a positive control for translation inhibition. H Yeast cells were incubated with L-AHA to label newly synthesized proteins and treated with 25 µM NT-2 toxin, 0.25% DMSO (CTR), or 50 µg/ml CHX. Mean fluorescence after click chemistry was quantified by flow cytometry; unlabeled cells indicate background signal. I Cell-free translation reactions with Renilla luciferase mRNA in S. cerevisiae lysate in the presence of 5 or 50 µM NT-2 or 1 mg/ml puromycin. The measured luminescence was normalized to the control reaction (CTR), which contained no supplement. Data are presented as mean ± SD values of three biological replicates (sets of translation reactions) averaged after three measurements.

Article Snippet: Cells were collected, washed with sterile water, and resuspended in SC medium lacking methionine but supplemented with L-AHA (TargetMol, Cat. No. T41090 ).

Techniques: Western Blot, Staining, Analysis, Positive Control, Phospho-proteomics, Inhibition, Control, Luciferase, Yeast, Incubation, Synthesized, Fluorescence, Flow Cytometry