Figure 1.
A genome-wide loss-of-function screen identified CCDC134 as an essential factor for TLR7/9 signaling. (A) Schematic of doxycycline-inducible MLKL-IRF5(122–498)-T2A-mCherry construct. (B) Schematic of TLR7-9 signaling in wildtype cells versus MLKL-IRF5(122–498)-T2A-mCherry expressing cells. (C) Representative dot-plot of FSC versus SSC gating used to assess cell viability (upper panel), with histogram for FSC and quantification of cell viability relative to the respective uninduced unstimulated control condition (lower panels). CAL-1 cells stably expressing MLKL-IRF5(122–498)-T2A-mCherry construct (CAL-1 reporter clone) and carrying sgRNA targeting SLC15A4 (sgSLC15A4) or control sgRNA targeting Renilla (sgRen) were induced or not with doxycycline (0.5 µg/ml) for 17 h and stimulated with R848 (2 µg/ml), CL307 (2 µg/ml) or CpG-B (ODN2006, 2 µM) for 6 h. unind.: uninduced; ind.: induced; unst.: unstimulated; Dox.: doxycycline. (D) Results of genome-wide loss-of-function screen in CAL-1 reporter cells (clone). Gene rank and gene score based on comparison between doxycycline-induced CL307 treated versus doxycycline-induced untreated conditions. (E) Cell viability of the indicated CAL-1 reporter cells assessed by flow cytometry (based on FSC versus SSC gating), relative to sgRen uninduced unstimulated (unind. unst.). Cells were induced or not with doxycycline (Dox.) (0.5 µg/ml, 17 h) before being stimulated or not with CL307 (2 µg/ml, 6 h). (F) Immunoblots of indicated CAL-1 reporter cells uninduced and stimulated with R848 (5 µg/ml, for 0–1 h). PhosTag, phos-Tag-containing gel. (G) Immunoblots of indicated knockout CAL-1 cells. (H) IL-6 (left panel) or IFNβ (right panel) production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (I–K) Immunoblots of indicated knockout CAL-1 cells stimulated with R848 (5 µg/ml, for 0–1 h) (I), CpG-B (ODN2006, 5 µM, for 0–3 h) (J) or TNFα (10 ng/ml, for 0–30 min) (K). In C, F, G, and I–K data are representative of two independent experiments. In E, data show mean ± SD of three independent experiments. In H, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: SourceData F1.

A genome-wide loss-of-function screen identified CCDC134 as an essential factor for TLR7/9 signaling. (A) Schematic of doxycycline-inducible MLKL-IRF5(122–498)-T2A-mCherry construct. (B) Schematic of TLR7-9 signaling in wildtype cells versus MLKL-IRF5(122–498)-T2A-mCherry expressing cells. (C) Representative dot-plot of FSC versus SSC gating used to assess cell viability (upper panel), with histogram for FSC and quantification of cell viability relative to the respective uninduced unstimulated control condition (lower panels). CAL-1 cells stably expressing MLKL-IRF5(122–498)-T2A-mCherry construct (CAL-1 reporter clone) and carrying sgRNA targeting SLC15A4 (sgSLC15A4) or control sgRNA targeting Renilla (sgRen) were induced or not with doxycycline (0.5 µg/ml) for 17 h and stimulated with R848 (2 µg/ml), CL307 (2 µg/ml) or CpG-B (ODN2006, 2 µM) for 6 h. unind.: uninduced; ind.: induced; unst.: unstimulated; Dox.: doxycycline. (D) Results of genome-wide loss-of-function screen in CAL-1 reporter cells (clone). Gene rank and gene score based on comparison between doxycycline-induced CL307 treated versus doxycycline-induced untreated conditions. (E) Cell viability of the indicated CAL-1 reporter cells assessed by flow cytometry (based on FSC versus SSC gating), relative to sgRen uninduced unstimulated (unind. unst.). Cells were induced or not with doxycycline (Dox.) (0.5 µg/ml, 17 h) before being stimulated or not with CL307 (2 µg/ml, 6 h). (F) Immunoblots of indicated CAL-1 reporter cells uninduced and stimulated with R848 (5 µg/ml, for 0–1 h). PhosTag, phos-Tag-containing gel. (G) Immunoblots of indicated knockout CAL-1 cells. (H) IL-6 (left panel) or IFNβ (right panel) production of indicated CAL-1 cells stimulated for 24 h with R848 (5 μg/ml). (I–K) Immunoblots of indicated knockout CAL-1 cells stimulated with R848 (5 µg/ml, for 0–1 h) (I), CpG-B (ODN2006, 5 µM, for 0–3 h) (J) or TNFα (10 ng/ml, for 0–30 min) (K). In C, F, G, and I–K data are representative of two independent experiments. In E, data show mean ± SD of three independent experiments. In H, data show mean ± SD of three stimulation replicates from one experiment representative of three independent experiments. Source data are available for this figure: SourceData F1.

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