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First published online 5 February 2008
doi: 10.1242/jcs.021352

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Ku is involved in cell growth, DNA replication and G1-S transition

McGill Cancer Centre and Department of Biochemistry, McGill University, Montreal, Quebec H3G 1Y6, Canada

View larger version (56K): [in this window] [in a new window]   Fig. 1. Reduced cell growth upon RNAi knockdown of Ku80. (A) HeLa cells treated with either transfection agent (control), a Ku80-targeting siRNA (Ku80-siRNA) or an siRNA with scrambled sequence (scr-siRNA) were grown for 24, 48, 72 and 96 hours. Untreated cells were also included as a control. Whole cell extracts were prepared at each time point and immunoblotted for Ku80. Actin was used as a loading control. (B) Staining of DNA with DAPI (blue) and anti-Ku80 (red) by immunofluorescence 96 hours after transfection with scr-siRNA or Ku80-siRNA. (C) Quantification of Ku80 levels in each sample shown in A. Values are expressed as relative optical density (ROD) compared to the untreated cells and represent the average of three experiments and one standard deviation (s.d). (D) Representative phase-contrast microscopic images of control, scr-siRNA-transfected and Ku80-siRNA-transfected cells at the indicated times after transfection. (E) Growth curves of the cells shown in D during the time-course of the Ku80 RNAi knockdown. Values represent the mean number of cells and the error bars are equivalent to 1 s.d.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Time-course of BrdU incorporation as a function of Ku80 knockdown. (A) Representative FACS analysis of BrdU incorporation of logarithmically growing control, scr-siRNA-transfected and Ku80-siRNA-transfected cells upon Ku80 silencing for 24, 72 and 96 hours. (B) Quantification of BrdU incorporation by each sample shown in A at the indicated times after transfection. Values are expressed as BrdU incorporation per 10,000 cells relative to control cells and are means of three individual experiments ± 1 s.d.

View larger version (33K): [in this window] [in a new window]   Fig. 3. Origin activation and DNA synthesis as a function of Ku80 knockdown. (A) Maps of the lamin B2 (left) and c-myc (right) origin loci, and melting curves of the primers corresponding to the origin-containing (LB2 and Myc11, respectively) and origin-lacking (LB2C and Myc1, respectively) amplicons. The location of the amplicons relative to the gene exons as well as their distance in kb is indicated on the maps. (B,C) Histogram plots of the lamin B2 and c-myc origin activities throughout the silencing time-course, as measured by nascent-DNA-strand abundance. The LB2 and Myc11 regions lie within the origins, whereas LB2C and Myc1 are distal origin-lacking regions. Values are expressed as ng of nascent DNA and represent three experiments ± 1 s.d. (D) Representative western blot analysis of PCNA protein expression at 72 hours post-transfection with Ku80 siRNA. Actin was used as a loading control. (E) Representative western blot analysis of the phosphorylation levels of the H2AX histone variant at 96 hours of Ku80 RNAi. As positive controls, extracts from cells treated with the drugs hydroxyurea (inhibitor of ribonucleotide reductase) and nocodazole (inhibitor of microtubule formation) were used. Immunostaining with a PanH3 antibody was used as a loading control.

View larger version (60K): [in this window] [in a new window]   Fig. 4. Expression and chromatin loading of DNA replication licensing factors. (A) Representative western blot analyses of Ku80, Orc1, Orc2, Orc3, Orc4, Orc6, MCM7 and Cdc45 protein levels in whole cell extracts prepared from untreated, control, scr-siRNA-transfected and Ku80-siRNA-transfected HeLa cells at 72 hours post-transfection. Actin was used as a loading control. (B) Relative optical densities (ROD) of Ku80, Orc1, Orc2, Orc3, Orc4, Orc6, MCM7 and Cdc45 immunoreactivities. Values are expressed as ratios of the individual RODs to the one corresponding to untreated cells, and the error bars represent 1 s.d. of three experiments. (C) Representative western blot analyses of Ku80, Orc1, Orc2, Orc3, Orc4, Orc6, MCM7 and Cdc45 protein levels in the chromatin-bound fraction prepared from untreated, control, scr-siRNA-transfected and Ku80-siRNA-transfected HeLa cells 72 hours post-transfection. Orc2, the association of which with chromatin is essentially the same throughout the cell cycle, was used as loading control. (D) Quantification of the immunoreactivity of the proteins shown in C after Ku80 knockdown for 72 hours. Signal intensities (RODs) are means ± 1 s.d.

View larger version (38K): [in this window] [in a new window]   Fig. 5. Cell-cycle analysis of Ku80-knockdown cells. (A) Asynchronous cultures of control, scr-siRNA-transfected and Ku80-siRNA-transfected HeLa cells were harvested after 96 hours of Ku80 silencing and stained with propidium iodide for monitoring of their cell-cycle distribution. Representative flow cytometric analysis is shown (top). (Bottom) The table shows the means (n=3) of the quantification of the percentage of cells present in each phase of the cell cycle. In brackets, 1 s.d. of three individual experiments is shown. (B) Western blot analysis of the cyclin A, cyclin D1 and cyclin E protein levels in whole cell extracts prepared from untreated, control, scr-siRNA-transfected and Ku80-siRNA-transfected HeLa cells. (C) Quantification of the relative optical densities of cyclin A, cyclin D1 and cyclin E. Values are expressed as the average of three experiments ± 1 s.d. (D) Schematic presentation of the cyclin/Cdk cycle and the various checkpoints. Silencing of Ku80 blocks the cells at the G1-S transition point, as evidenced by high levels of cyclin E (dark-orange arrow).

View larger version (51K): [in this window] [in a new window]   Fig. 6. Activation of a replication-stress checkpoint and analysis of apoptosis and ssDNA levels. (A) Protein expression of the cell-cycle regulators Chk1-P, Chk2-P, Cdc25A, p21 and Cdk2 in whole cell extracts prepared at 96 hours from untreated, control, scr-siRNA-transfected and Ku80-siRNA-transfected HeLa cells. Actin was used as loading control. Extracts from cells treated with the drugs hydroxyurea (10 mM) and nocodazole (100 ng/µl) were used as positive controls for the activational phosphorylation of Chk1 and Chk2. (B) Quantification of the protein levels of the cell-cycle regulators shown in A. Values represent the average of three experiments and the error bars correspond to 1 s.d. Chk1-P levels were not included in the bar graph because no detectable levels were observed. (C) Protein expression of the Cdk2 regulators Cdc25A, p21 and Cdk2 in whole cell extracts prepared after Ku80-siRNA transfection in HCT-116 Ku80+/– cells. (D) Genomic DNA was isolated from untreated, control, scr-siRNA-transfected and Ku80-siRNA-transfected HeLa cells after 96 hours of Ku80 silencing and was subjected to electrophoresis in order to determine its integrity. Extracts from cells treated with the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA), which is known to induce apoptosis, were used as positive controls. (E) Representative western blot analysis of the protein levels of full-length PARP-1 and the full-length precursor form of caspase 3, which are cleaved during apoptosis. (F) Chromatin association of the ssDNA-binding protein RPA70 at 96 hours post-transfection.