Validation of candidate genes involved in typhoid toxin intoxication.

(A) Genotyping of the CRISPR/Cas9-generated knockout cells. Genomic DNA was purified from CRISPR-Cas9 edited HEK293T cell lines and analyzed by PCR with specific primers listed in S3 Table. The TMED2-deficient cell line was examined by Western blot with an anti TEMED2-specific antibody. Dotted lines indicate places where the experimentally relevant lanes were spliced together (all lanes originate from a single gel). (B) Relative toxicity of typhoid toxin in the indicated knockout cell lines after treatment with a serial dilution of purified toxin. The relative toxicity was determined from the percentage of cells at the G2M phase fitted by nonlinear regression as indicated in the Materials and Methods. Values, which were normalized relative to wild type (considered 100) are the mean ± SEM of independent determinations. ****p < 0.0001; n. s.: differences not statistically significant; two-tailed Student’s t-test.