Fig. 1

PB transposon mutagenesis screen. (A) Scheme of the PB mutagenesis screening process. Parental cancer cells were transfected with transposition (PB) and transposase (PBase) plasmids, passaged with puromycin to generate mutagenized libraries, and treated with PLX4720 to generate resistant colonies or pools. Insert sites were detected using linker-mediated PCR and NGS, with the shown amplicon structure of the transposon (PB), host genomic DNA (gDNA), linker, and PCR primers (arrows). (B) The PB transposition plasmid includes a cassette with an antibiotic selection marker (puroR) for transposon-mutagenized cell maintenance, a CMV promoter (CMV) and a splice donor (SD) for host gene transcription and alternative splicing, and two inverted repeats (IRs) for transposition. (C) Clonogenic assays showing the sensitive parental cells and PLX4720-resistant cells from transposon screen. (D) High confidence hits identified from resistant pools are displayed by genomic location (x-axis: chrs.1–22,x). The inserts for each gene were summarized, read numbers of each gene normalized to the total number of reads were plotted as y-coordinates, and the dot sizes represent the numbers of insertion events. Genes with a predicted activation effect are shown as orange bubbles, and candidates for follow-up validation assays are circled and labeled.