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. Author manuscript; available in PMC: 2019 Jan 30.
Published in final edited form as: Cell Rep. 2018 Jan 30;22(5):1185–1199. doi: 10.1016/j.celrep.2018.01.022

Figure 6. RB-Deficient Cells Are Prone to Replicative Catastrophe and Mitotic Dysregulation.

Figure 6

(A) Flow-cytometry traces of MB231 and MB231 RB CRISPR cells treated with the indicated agents (x axis, DNA content; y axis, BrdU incorporation). The positions of 2N, 4N, and 8N ploidy are indicated.

(B) Quantitation of BrdU intensity in control (red) or CHK inhibitor (CHIR124)-treated (blue) cells. The mean and SD are shown. The BrdU collapse is indicative of increased S phase DNA damage and failure or replication.

(C) Quantitation of the percentage of BrdU-positive cells treated with CHIR124. The mean and SD are shown. Significant differences between RB-proficient and deficient cells were determined by t test (**p < 0.01).

(D) Quantitation of cell ploidy in the RB-proficient and deficient cells with the indicated treatments. The mean and SD are shown. Significant differences between RB-proficient and deficient cells were determined by t test (**p < 0.01).

(E) Quantitation of chromatin-associated PCNA following treatment with AZD7762. The mean and SD are shown (**p < 0.01). Representative immunofluorescence images of RB-proficient and deficient cells following treatment with the CHK inhibitor AZD7762 (scale bar, 25 μm).

(F) Immunoblotting of the indicated cell lines treated with increasing doses of CHK inhibitor (AZD7762). PARP cleavage is a marker for apoptotic cell death.

(G) MB436 cells either transduced with control virus (LacZ) or expressing constitutively active RB (7LP) were treated with volasertib, and the percentage of cells exhibiting mitotic entry was determined by staining for phosphorylated serine 10 of histone H3. The mean and SD are shown. Significant effect of constitutively active RB on the response to volasertib was determined by t test (***p < 0.001). Representative images showing combined staining for EdU and phosphorylated serine 10 histone H3 and DAPI (scale bar, 20 μm).

(H) MB436 cells either transduced with control virus (LacZ) or expressing constitutively active RB (7LP) were treated with CHIR124, and the percentage of cells exhibiting γ-H2AX reactivity was determined by immunofluorescent staining. The mean and SD are shown. Significant effect of constitutively active RB on the response to CHIR124 was determined by t test (***p < 0.001). Representative images showing combined staining for EdU and phosphorylated γ-H2AX and DAPI (scale bar, 20 μm).