1. Chk1 and 14‐3‐3 proteins inhibit atypical E2Fs to prevent a permanent cell cycle arrest
ASchematic 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. BFACS 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. CTranscript 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, EImmunofluorescence 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.
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EMBO J, Volume: 37, Issue: 5, First published: 23 January 2018, DOI: ( /embj )