American Society of Hematology

Figure 4

$Figure 4. Cotreatment with MLN4924 and belinostat disrupts the intra-S-phase checkpoint and HR or NHEJ repairs. (A) MV-4-11 cells were treated with 200 nM PXD-101 ± 100 nM MLN4924 for 16 hours, after which cells were pulse-labeled with EdU for 30 minutes, followed by double staining for EdU and F-actin (Alexa Fluor phalloidin) with DAPI counterstaining. (Top panels) Left, DAPI (blue); middle, EdU (green); right, dot plot of FCM determining the percentage of EdU-positive cells (inset, histogram). (Bottom panels) Left, EdU and phalloidin (red) merged; middle, EdU and DAPI merged; right, EdU, phalloidin, and DAPI merged. Scale bars, 20 μm. (B) After exposure to 300 nM PXD-101 ± 50 to 100 nM MLN4924 for 16 hours, whole-cell lysates (WCL) and nuclear (Nuc) or cytosolic (Cyto) fractions of U937 cells were prepared and subjected to western blot analysis using the indicated antibodies. (C) MV-4-11 and U937 cells were treated as described in panels A and B, respectively, after which western blot analysis was performed to assess BRCA1 phosphorylation (S1524, S1423) and FANCD2 ubiquitination (S, an inactive unubiquitinated isoform; L, an active monoubiquitinated isoform). (D) U937 cells were incubated with 300 nM MLN4924 ± 100 nM PXD-101 for 16 hours, followed by double staining for 53BP1 (red) and γH2A.X (green) with DAPI counterstaining. Scale bars, 10 μm. (E) U937 cells were treated with 300 nM PXD-101 ± 100 nM for 16 hours, after which chromosome-spreading analysis (DAPI staining) was carried out to monitor chromosome morphology and pulverization, characterized by fragments of DAPI staining material that were clustered into a discrete location on the spread. Scale bars, 10 μm.$

Cotreatment with MLN4924 and belinostat disrupts the intra-S-phase checkpoint and HR or NHEJ repairs. (A) MV-4-11 cells were treated with 200 nM PXD-101 ± 100 nM MLN4924 for 16 hours, after which cells were pulse-labeled with EdU for 30 minutes, followed by double staining for EdU and F-actin (Alexa Fluor phalloidin) with DAPI counterstaining. (Top panels) Left, DAPI (blue); middle, EdU (green); right, dot plot of FCM determining the percentage of EdU-positive cells (inset, histogram). (Bottom panels) Left, EdU and phalloidin (red) merged; middle, EdU and DAPI merged; right, EdU, phalloidin, and DAPI merged. Scale bars, 20 μm. (B) After exposure to 300 nM PXD-101 ± 50 to 100 nM MLN4924 for 16 hours, whole-cell lysates (WCL) and nuclear (Nuc) or cytosolic (Cyto) fractions of U937 cells were prepared and subjected to western blot analysis using the indicated antibodies. (C) MV-4-11 and U937 cells were treated as described in panels A and B, respectively, after which western blot analysis was performed to assess BRCA1 phosphorylation (S1524, S1423) and FANCD2 ubiquitination (S, an inactive unubiquitinated isoform; L, an active monoubiquitinated isoform). (D) U937 cells were incubated with 300 nM MLN4924 ± 100 nM PXD-101 for 16 hours, followed by double staining for 53BP1 (red) and γH2A.X (green) with DAPI counterstaining. Scale bars, 10 μm. (E) U937 cells were treated with 300 nM PXD-101 ± 100 nM for 16 hours, after which chromosome-spreading analysis (DAPI staining) was carried out to monitor chromosome morphology and pulverization, characterized by fragments of DAPI staining material that were clustered into a discrete location on the spread. Scale bars, 10 μm.

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