Fig. 2
Haploid genetic screens in KBM7 FADD- cells identify genes required for necroptosis. a–c Circos plots of haploid genetic screens in KBM7 FADD- cells with necroptosis induction by 10 µM SMAC mimetic birinapant (a) 100 ng/ml TNFα (b) and 1 µM SMAC mimetic and 100 ng/ml TNFα combined (c). Each dot represents a mutagenized gene identified in the resistant cell population, dot size corresponds to the number of independent insertions identified for each gene and distance from center indicates the significance of enrichment compared to an unselected control data set. Hits with an adjusted p-value < 10‒10 are labeled. d Bubble plot depicting the top hits over all three screens ranked according to adjusted p-value in the TNFα screen. Bubble size corresponds to the number of independent insertions identified and color gradient reflects the significance of enrichment. e, f Multi-color competition assay (MCA) of KBM7 FADD- SpCas9 cells transduced with a GFP marker (GFP+) and sgRNAs targeting either SLC39A7 or RIPK1 (e), SP1 or TNFR2 (f), or Renilla luciferase (sgRen) as control, against cells transduced with sgRen and an mCherry marker (mCherry+). The cell populations were mixed at 1:1 ratio, treated with SMAC mimetic (1 µM) or TNFα (10 ng/ml), and analyzed after 14 days by flow cytometry. Data represent mean value ± s.d. of two independent experiments performed in duplicates, n.d. (not determined) indicates wells with no outgrowth
