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. 2017 Jun 12;36(14):2161–2176. doi: 10.15252/embj.201696082

Figure 8. Kap1 is an ATM/Wip1 target on chromatin.

Figure 8

  1. U2OS cells transfected with GAPDH or Wip1 siRNA were treated with NCS (5 nM) and collected after 2, 6, and 20 h. Chromatin fractions were probed with indicated antibodies.
  2. RPE cells transfected with Wip1 siRNA were microirradiated, fixed 1 or 24 h later, and stained with the indicated antibodies.
  3. HA‐KAP1‐WT or HA‐KAP1‐S824A were immunopurified from cells exposed to NCS, incubated with His‐Wip1 and probed with pS824‐KAP1 or KAP1 antibody.
  4. U2OS cells were fixed 1 h after treatment with NCS, incubated with His‐Wip1 (0–5 ng/μl) and probed for γH2AX and pS824‐KAP1. Plot shows mean nuclear fluorescence intensity of at least 100 cells per condition; error bars indicate SD.
  5. Kap1 is dephosphorylated before Plk1 activation. RPE cells transfected with TP53 siRNA and U2OS cells were followed as in Fig 4A and stained for pS824‐Kap1. G2 cells (0 h, blue), G2‐arrested cells without detectable Plk1 activity (5 h, red), and recovering G2 cells with increasing Plk1 activity (30 h, green). For RPE cells, the times were modified as indicated. Black bars indicate median and circles correspond to individual cells.
  6. RPE cells transfected with GAPDH or TP53 siRNA were synchronized by HU, released to fresh media for 5 h (R5), and treated with NCS for indicated times. Nocodazole (NZ) was added 1 h after NCS. Where indicated, cells were incubated in the presence of BI2536. Whole‐cell lysates were probed with indicated antibodies. Antibodies against active forms of Plk1 and Aurora A kinase and to a mitotic marker pS10‐H3 were used to determine when cells recover from the checkpoint arrest.
  7. Overexpression of Kap1‐S824A phenocopies ATM inhibition. Cumulative mitotic entry of ≥ 300 U2OS cells expressing inducible HA‐tagged Kap1‐wt (red) or Kap1‐S824A (green) after treatment with NCS (4 nM) and subsequent treatment after 1 h with ATRi or ATRi + ATMi.