Figure 3. Chk1 inhibits the transcriptional repressor function of E2F7 and E2F8 to promote cell cycle progression and prevent apoptosis.

• A
  Left panel shows a schematic view of the experimental setting. HeLa/TO cells were synchronized with hydroxyurea (HU) for 16 h, then released by washing and adding new medium containing doxycycline (Dox) to induce expression of the transgenes. Cell lines incubated with medium without doxycycline (Vehicle, Veh) were used as controls. Expression of WT and Chk1 mutant versions of E2F7/8 was monitored by EGFP fluorescence, DNA was visualized by adding fluorescent SiR‐DNA to the medium, and cell morphology was evaluated by differential interference contrast (DIC). Per condition, 100 individual cells were traced. Each cell was followed until it successfully finished mitosis and divided into two daughter cells, for a maximum of 24 h. The graphs on the right show the quantification of the mitotic events during live cell imaging. Scale bar: 10 μm.
• B
  Quantification of apoptosis by flow cytometric analysis of Annexin‐V staining. Experiments were performed as outlined in (A), and cells were harvested for flow cytometry 24 h after HU release. Apoptotic cells were counted as Annexin‐positive and DAPI‐negative.
• C, D
  Quantitative PCR of CDC6, RAD51, CDC25A, and CCNE1 expression in HeLa/TO cell lines expressing wild‐type and mutant versions of E2F7/8.
• E
  Chk1 phosphorylation does not change the promoter enrichment of E2F7 and E2F8. HEK cells were transfected with either PEI reagent alone (control) or indicated plasmids tagged with GFP. 48 h after transfection, cells were harvested for chromatin immunoprecipitation (ChIP) followed by qPCR. Histogram represents the enrichment ratio (bound/input) in E2Fs target gene promoters.

Data information: In (B–D), data represent average ± SEM (n = 3); *P < 0.05 or **P < 0.01 (Student's t‐test in B, and two‐way ANOVA in C and D).