Belinostat attenuates activation of the ATR/Chk1/Wee1-mediated DNA damage checkpoint pathway by MLN4924. (A-C) Cells were treated (24 hours) with PXD-101 (U937, 300 nM; MV-4-11 and MOLM-13, 200 nM) ± 100 nM MLN4924, after which western blot analysis was performed to monitor expression of Cdt1 (top band, *nonspecific bands), total and/or phosphorylated ATR (S428), Chk1, Wee1 (S642), H2A.X (S139). (D) MV-4-11 cells were treated with 200 nM PXD-101 ± 100 nM MLN4924 for 16 hours, after which cytospin slides were prepared and stained with the primary antibodies against p-Chk1 (S345) and γH2A.X, followed by Alexa Fluor 488– or Alexa Fluor 594–conjugated secondary antibody. Images (×64) were captured using a Zeiss LSM 700 confocal laser-scanning microscope. Arrow, Cells containing γH2A.X foci with no or few p-Chk1 colocalization foci; arrowhead, apoptotic cells. (Top panels) Left, γH2A.X (red); middle, p-Chk1 (green); right, γH2A.X and p-Chk1 merged. (Bottom panels) Left, γH2A.X and DAPI merged; middle, p-Chk1 and DAPI merged; right, DAPI (blue). Scale bars, 10 μm. Squares indicate areas corresponding to images with higher magnification as shown in supplemental Figure 3F. (E) U937 cells were stably transfected with constructs encoding shRNA targeting human CHEK1/Chk1 (shChk1), Wee1 (shWee1), or scrambled sequence (shNC). Cells were then treated with the indicated concentrations of MLN4924 for 24 hours, after which western blot analysis was performed to monitor expression of Chk1, Wee1, and γH2A.X.