Effects of double knockdowns of ASCC3 and genes involved in translation quality control on PF8503 toxicity.

(A) Lentiviral construct used to generate double knockdown cell lines. For each construct, the sgRNA targeting ASCC3 was placed after the human U6 (hU6) promoter, and a second sgRNA (scrambled or targeting ASCC2, NEMF, or HBS1L) was placed after the murine U6 (mU6) promoter. (B) PF8503 phenotype (Rho) obtained in competitive growth assays performed using double knockdown cell lines (grey), compared with the rho phenotype expected from the sum of phenotypes of individual knockdowns. Due to less efficient knockdown of ASCC3 in the dual-sgRNA context, the individual phenotype of ASCC3 knockdown was taken from the cell line expressing mU6-scrambled sgRNA-hU6-ASCC3 sgRNA pair. All other phenotypes were taken from the individual knockdowns in Fig 2A. Experiments were carried out in biological triplicate for ASCC3-NEMF cell line and in 6 replicates for other cell lines, with mean and standard deviation shown.