Figure 1. Modulation of the cellular phosphoproteome following NCS treatment.

• A
  Schematic workflow of the phosphoproteomic experiment. Cells were treated with 20 ng/ml NCS in the presence of selective inhibitors of ATM (KU60019, 5 µM), ATR (AZ20, 0.5 µM), DNA‐PK (NU7441, 5 µM), or DMSO (inhibitor solvent). Inhibitors were added 30 min prior to NCS treatment, and samples were collected 20, 60, and 240 min following NCS addition. Protein extracts were digested into peptides and subsequently enriched for phosphopeptides, which were measured using LC‐MS/MS and subjected to label‐free quantification and subsequent data processing and analysis using the MaxQuant (Cox & Mann, 2008; Tyanova et al, 2016a) and the Perseus (Tyanova et al, 2016b) software. An FDR of 0.05 together with a minimal fold change of twofold was applied to determine regulation in response to NCS and PIKK inhibitors.
• B
  Enriched GO cellular compartments (gray) among NCS‐induced phosphorylations and dephosphorylations, and GO biological processes (purple‐red) among NCS‐induced phosphorylations, in WT cells. Enrichment was tested using the Fisher exact test implemented in Perseus (Tyanova et al, 2016b).
• C
  Enriched motifs among NCS‐induced phosphorylations (yellow) and dephosphorylations (gray) in WT cells. Enrichment was tested using the Fisher exact test implemented in Perseus (Tyanova et al, 2016b).