Validation of MLKL-IRF5(122–498) reporter cell line and genome-wide CRISPR/Cas9 screening. (A) Schematic of MLKL-IRF5(122–498) construct. DBD: DNA-binding domain; LK: linker region; IAD: IRF association domain; AR: auto-inhibitory region; NBB: N-terminal bundle and brace; PKD: pseudo kinase domain. (B) Representative dot-plot of FSC versus SSC gating used to assess cell viability (upper panel) with histogram for FSC (left lower panel), quantification of cell viability relative to the respective unstimulated (unst.) condition (middle lower panel) and ratio of mCherry/GFP gMFI relative to sgRen unstimulated (unst.) cells (right lower panel). CAL-1 cells stably expressing MLKL-IRF5(122–498)-T2A-mCherry construct (population) and indicated knockout were induced with doxycycline (0.5 µg/ml) for 17 h before being stimulated or not with R848 (5 µg/ml) for 6 h. (C) Representative histogram of mCherry/GFP gMFI ratio (relative to sgRen uninduced unstimulated [unind. unst.] condition) of CAL-1 reporter cells (clone) and indicated knockout. Cells were induced or not with doxycycline (Dox.) (0.5 µg/ml) for 17 h before being stimulated or not with R848 (2 µg/ml), CL307 (2 µg/ml) or CpG-B (ODN2006, 2 µM) for 6 h. (D) Immunoblots of CAL-1 reporter cells (clone) induced or not with doxycycline (Dox.) (0.5 µg/ml) for 17 h before being stimulated with R848 (5 µg/ml, for 0–1 h). Unlike the anti-phospho-IRF5 antibody, the IRF5 antibody used detects endogenous IRF5 but not the MLKL-IRF5(122–498)-T2A-mCherry construct. Red arrow indicates MLKL-IRF5(122–498) construct, black arrow endogenous IRF5 or MLKL and asterisk a non-specific band. (E) Representative dot-plot gating used to assess cell viability (left panel) and quantification of cell viability relative to uninduced untreated (unind. untr.) condition (right panel). CAL-1 cells stably expressing MLKL-IRF5(122–498)-T2A-mCherry construct (clone) were induced with doxycycline (Dox.) (0.5 µg/ml) and simultaneously treated or not with Z-VAD-FMK (Z-VAD) (20 µM) or NSA (5 µM) for 17 h before being stimulated or not with R848 (2 µg/ml) for 6 h. Small debris and cell aggregates were neglected using FSC and SSC gating while dead cells were excluded by gating on the negative/low population (Near-IR Live/Dead [L/D]). na: condition not assessed. (F) Schematic of genome-wide loss-of-function screen. (G) Venn diagram showing the overlap of hits with a fold change >1.55 for each of the indicated comparisons (left panel), and a list of the 29 overlapping hits in the different comparisons including the induced CL307 condition (right panel). The ranking, indicated in bracket, is based on the induced CL307 versus induced untreated comparison. (H) Cell viability quantification of an extended panel of CAL-1 reporter (clone) knockout cell lines of one independent experiment previously illustrated in Fig. 1 E. Cells were induced or not by doxycycline (Dox.) (0.5 µg/ml) for 17 h before being stimulated or not with CL307 (2 µg/ml) for 6 h. Data are representative of two (B–D) or one (H) independent experiments. In E (right panel), data show mean ± SD from three independent experiments. Source data are available for this figure: SourceData FS1.