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. 2022 Sep 20;41(21):e110372. doi: 10.15252/embj.2021110372

Figure 1. Genome‐wide siRNA screening.

Figure 1

  • A
    Schematic diagram of the two consecutive siRNA screens. NF‐κB‐driven luciferase activity served as a readout for pathway activation. The primary screen was performed using a genome‐wide siRNA library. A total of 320 candidates were selected for a secondary counter screen with TNFα to narrow down DNA damage‐specific regulators of the NF‐κB pathway. Responsiveness of the reporter cell line to NF‐κB activating stimuli was analyzed by western blotting (Fig EV1A).
  • B
    Cumulative distribution of Z‐scores of the genome‐wide siRNA screening. Candidates with low Z‐scores are putative positive regulators as their depletion abrogated NF‐κB activation by DNA damage (red circle). Putative negative regulators with high Z‐scores exhibited elevated NF‐κB activity (blue circle).
  • C
    Z‐scores of the best‐scoring siRNAs for selected previously known and newly identified pathway regulators. Hits include VPS28 (Mamińska et al, 2016), CYLD (Trompouki et al, 2003), NFKBIA (Haskill et al, 1991), TRAF3 (Vallabhapurapu et al, 2008), N4BP1 (Shi et al, 2021), TANK (Wang et al, 2015), SENP1 (Lee et al, 2011; Shao et al, 2015), PARG (Cortes et al, 2004), RELA (Li et al, 2001), TIFA (Fu et al, 2018), CHUK (Colomer et al, 2019), TRAF6 (Hinz et al, 2010) and IKBKG (Mabb et al, 2006) for expected factors.
  • D
    Representative relative luciferase units (RLU) of the differential counter screen of the 320 preselected hits are shown. The RLU of hits is displayed for etoposide and TNFα screens (left and right panels). See Dataset EV4 for details. RLU levels of treatment controls are indicated with red lines. Hits that abrogated etoposide‐induced NF‐κB activity were classified as group I. Hits in the TNFα counter screen with higher activity than in siTRAF6‐transfected cells constituted group II. The intersection of the two groups indicates putative DNA damage‐selective positive regulators. Note that TNFα may trigger secondary NF‐κB activation events through autocrine/paracrine mechanisms. See Dataset EV4 for luminescence values.