CHO cells do not require <i>de novo</i> mRNA synthesis during late G₁, or for progression through S-phase.

<p>(<i>A</i>) Diagram illustrating the experimental design for the data obtained in <i>B&C</i>. (<i>B&C</i>) CHO cells were synchronized in G₀ by isoleucine deprivation and then released into the cell cycle by re-addition of complete medium. At the times indicated, control cells were pulsed with BrdU to determine the kinetics of progression through G₁ into S-phase (gray columns on left in <i>C</i>; examples shown in <i>B</i>). Cells treated with 50 µM DRB at the times indicated were allowed to progress to the peak of S-phase at 12 hrs, at which time they were pulsed with BrdU to determine the percentage of cells that could enter S-phase following different times of DRB exposure (black columns on right in <i>C</i>; examples shown in <i>B</i>). As a control, the DMSO carrier was added to a parallel culture at 1 hr and remained until the BrdU pulse at 12 hrs (white column on right in <i>C</i>). The 12 hr untreated control (gray column on right in <i>C</i>) indicates the maximum number of BrdU-labeled cells obtained without drug treatment. The means of triplicate counts of ∼200 cells/field+/−1 s. d. are shown. (<i>D</i>) RT-PCR analysis of c-<i>myc</i> mRNA levels on samples collected at the indicated times, with and without DRB exposure at 8 hrs. (<i>E</i>) Synchronized CHO cells were untreated (control, top row), or treated with 50 µM DRB at 8 hrs (bottom row), and pulsed with BrdU at each time point indicated in order to measure progression into and through S-phase. At least three fields of ∼200 cells were scored, and averages are displayed in panels with representative fields. Standard deviations (not shown) were within 1–5% for all panels. (<i>F</i>) Parallel to the samples in <i>E</i>, cells were collected and processed by flow cytometry using PI staining.</p

MCM, Cdc45, and PCNA load in the final 3 hrs of G₁ in CHO cells.

<p>(<i>A</i>) Parallel to the BrdU and flow cytometry collection in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0005462#pone-0005462-g003" target="_blank">Figure 3 E&F</a>, CHO cells (half treated with DRB at 8 hrs) were collected and separated into total cell lysates (TCE), or fractionated into nucleosolic/cytosolic detergent-soluble extracts (S1) or chromatin-bound detergent-resistant extracts (P3). Immunoblotting with the indicated antibodies was performed on lysates from equal cell numbers loaded into each lane. The G₁-S transition in CHO cells (9 hrs after release) is overlayed in gray. (<i>B</i>) An enlargement of the time points from part <i>A</i> for hours G₀ through 9 is shown for Mcm2 and Mcm5 immunoblots.</p

Summary of results described in this report.

<p>Mammalian cells require ongoing mRNA synthesis in the first part of G₁-phase. Concurrent with this timeframe, the Rb protein is hypophosphorylated and MCMs have not loaded onto chromatin at the preRCs. In the final 3–4 hours of G₁-phase, mammalian cells pass through a transition when Rb is hyperphosphorylated, MCMs load onto chromatin, and new mRNA synthesis is no longer rate-limiting for MCM, Cdc45, or PCNA loading, nor for the eventual progression of the cells into and through S-phase.</p

<i>De novo</i> mRNA synthesis is not required in the final 3–4 hrs of G₁ for entry into S-phase.

<p>(<i>A</i>) Diagram illustrating the experimental design for the data obtained in <i>B</i>. (<i>B</i>) Balb/MK cells were synchronized in G₀ by EGF deprivation and then released into the cell cycle by re-addition of EGF. At the times indicated, control cells were pulsed with BrdU to determine the kinetics of progression through G₁ into S-phase (gray columns on left). Cells treated with 50 µM DRB at the times indicated were allowed to progress to the peak of S-phase at 15 hrs, at which time they were pulsed with BrdU to determine the percentage of cells that could enter S-phase following different times of DRB exposure (black columns on right). As a control, the DMSO carrier was added to a parallel culture at 1 hr and remained until the BrdU pulse at 15 hrs (white column on right). The 15 hr untreated control (gray column on right) indicates the maximum number of BrdU-labeled cells obtained. The means of triplicate counts of ∼200 cells/field+/−1 s. d. are shown. <i>C</i>) Asynchronous, logarithmically growing human MCF7 cells were treated with 50 µM DRB from time zero (squares), or not treated (circles), during a 24 hr period. BrdU was added at the beginning of the experiment and remained throughout the 24 hr period. At the indicated times, samples were fixed and processed for BrdU incorporation to determine the percentage of cells that had entered S-phase. The first time point was exposed to BrdU for 30 min before fixation. The means of triplicate counts of ∼250 cells/field+/−1 s. d. are shown.</p

MCM, Cdc45, and PCNA load in the final 4 hrs of G₁ in Balb/MK cells.

<p>(<i>A</i>) BrdU was pulsed into MK cells at the indicated times following release from quiescence to determine the kinetics of synchronization and entry into S-phase. (<i>B</i>) In parallel with the BrdU-pulsed samples in A, MK cells were collected at the indicated times and separated into total cell lysates (TCE), or fractionated into nucleosolic/cytosolic detergent-soluble extracts (S1) or chromatin-bound detergent-resistant extracts (P3). Immunoblotting with the indicated antibodies was performed on lysates from equal cell numbers loaded into each lane. The G₁-S transition in MK cells (12 hrs after release) is overlayed in gray.</p