Fig 2: (A) BRG1 depletion attenuates UV-induced ATR activation and phosphorylation of ATR target Chk1. BRG1-depleted HeLa cells and control cells were exposed to 30 J/m2 and harvested 1 and 3 h after treatment. Cell lysates were separated on PAGE gels, followed by immunoblotting using anti-ATR, anti-phospho-ATR, anti-Chk1, anti-phospho-Chk1, and anti-Tubulin antibodies. ATM activation was also examined. (B) Two additional shRNAs targeting BRG1 confirms the effect of BRG1 on ATR activation. (C) BRG1 is an upstream regulator of BRCA1 recruitment and ATR activation. BRG1 may facilitate BRCA1 recruitment to UV-stalled forks in replicating cells, through its chromatin remodel activity. In addition, BRG1 is upstream of ATR to modulate ATR activation and phosphorylation (p) of its downstream substrates, including RPA, BRCA1, and Chk1.

Fig 3: Model of ATM-KAP1-mediated regulation of EBV replication.i. KAP1 represses EBV lytic genes in latently infected cells. ii. Lytic cycle activation signals cause ATM-mediated phosphorylation of KAP1 at S824; phosphorylated KAP1 is unable to repress EBV lytic genes. These events allow expression of lytic genes of all kinetic classes in response to lytic cycle activation triggers. Events triggering the initial expression of ZEBRA and activation of ATM are unclear and shown by dashed arrows. Chloroquine, known to activate ATM, causes phosphorylation of KAP1 at S824 resulting in activation of EBV lytic cycle; IE: immediate early, E: early, L: late lytic genes.

Fig 4: Effect of PI3-kinase-like kinase (PIKK)- and CHK1/2 inhibitors on the proliferation of 8226-LR5 cells.The MTT assay was used to monitor the survival after 72h incubation with the various inhibitors ±2.5 Melphalan. CHK1/2 inhibitor AZD7762 (0.1 µM), DNA-PK inhibitor NU7441 (0.2 µM), ATM inhibitor KU55933 (3 µM), ATR inhibitor VE821 (1 µM). Whereas a moderate inhibition on proliferation was observed with either inhibitor alone, strong inhibition was observed in combination with melphalan for the CHK1/2, DNA-PK and ATR inhibitors. Each bar represents the mean of at least 5 independent experiments with standard deviations as indicated. P-values were calculated using 2-sample t-test with equal variance. >95%, >99% and >99.9% confidence levels indicated by *, **, and ***, respectively.

Fig 5: Endogenous ATM and KAP1 directly interact in situ exclusively in lytic cells.A. HH514-16 cells were treated with NaB or left untreated and harvested 24 hours later for immunoprecipitation with normal rabbit IgG, rabbit anti-ATM or rabbit anti-KAP1 antibody. Input samples and immunoprecipitated materials were subjected to immunoblotting with indicated antibodies; IgG hc: IgG heavy chain. B. HH514-16 cells were treated with NaB and harvested at 24 hour post-treatment for examination by proximity ligation assay (PLA) with antibodies to ATM and KAP1 (upper 2 panels), anti-ATM antibody and rabbit IgG (middle panel) or anti-KAP1 antibody and goat IgG (lower panel) and subsequent immunostaining with antibody against ZEBRA; arrows: fluorescent foci indicative of intermolecular interactions. Experiments were performed twice.