FIG 1.

Chk1 protein level is reduced in the nuclei of MRC5-SV cells depleted of Pol κ. (A) (Left) Western blot analysis of Chk1 and Pol κ in MRC5-SV nuclear extracts 48 h after transfection with a control (Luc), Pol κ (Polκ), or Chk1 (Chk1) siRNA. Cells were left untreated or treated with 1 mM HU for 1 h. ORC4 is shown as a protein-loading control. Quantification of Chk1 is relative to siLuc condition. (Right) Relative Chk1 or Pol κ protein levels in siPolκ nuclear extracts were normalized to the siLuc condition; data from 5 independent experiments in MRC-SV cells are plotted. The regression curve (dashed line) and R-square are shown. (B) MRC5-SV cells were cotransfected with the indicated siRNAs and a vector expressing either GFP-empty or GFP-Pol κ (GFP-Polκ). The fluorescence intensity of Chk1 was quantified in each nucleus. Medians with 25% and 75% interquartile ranges are represented. ***, P = 0.001; ****, P < 0.0001; Mann-Whitney test. A.U, arbitrary units; ns, not significant. (C) Western blot analysis of Pol κ, GFP-Pol κ, and Chk1 in nuclear extracts of MRC5-SV cells 48 h after cotransfection with a control siRNA (Luc) or targeting the 3′ UTR of Pol κ (Polκ3′) with a vector expressing either GFP-empty or GFP-Polκ. MCM7 is shown as a loading control. (D) Western blot analysis of nuclear extracts from MRC5-SV cells transfected with a control (Luc), Polκ, or Chk1 siRNA. Immunodetected proteins are indicated. Fibrillarin and ORC2 are shown as loading controls. (E) Relative Chk1 or Pol κ protein levels in siPol κ whole-cell extracts of MRC5-SV cells were normalized to the siLuc condition. Data from 9 independent experiments are plotted. The regression curve (dashed line) and R-square are shown.