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. 2018 Jan 23;37(5):e97877. doi: 10.15252/embj.201797877

Figure 4. Loss of Chk1 results in E2F7/8‐dependent cell cycle arrest and DNA damage.

Figure 4

  • A
    Schematic overview of the experimental setting and immunoblot analysis for the validation of the efficient siRNA‐mediated knockdown of Chk1, E2F7, and E2F8 at HU (0 h) or 8 h after HU release. Protein levels of atypical E2Fs targets RAD51 and CDC6 are shown. Detection of γ‐tubulin was used as loading control.
  • B
    FACS cell cycle profiles at 0, 4 and 8 h after HU release from propidium iodide‐stained HeLa cells incubated with siRNAs directed against Chk1 and E2F7/8. Scrambled (scr) siRNA was used as control.
  • C
    Transcript levels of E2F7/8 target genes CDC6, RAD51, CDC25A, and CCNE1 after HU treatment for the indicated siRNA groups measured by quantitative PCR. Fold changes were adjusted to scrambled condition.
  • D, E
    Immunofluorescence staining showing the intensities of Rad51 and γ‐H2AX in HeLa cells. Cells were transfected with indicated siRNA and incubated with HU for 16 h before harvest. Nuclear DNA was stained with DAPI. For quantification (right side), five fields of 40× magnification pictures were selected randomly and subjected to ImageJ software analysis. Scale bars: 20 μm.
Data information: In (C), data represent average ± SEM (n = 3); in (D, E), bars represent average (n = 150); *P < 0.05 or **P < 0.01 (Student's t‐test) or n.s. for not significant.Source data are available online for this figure.