Restriction point and metabolic checkpoint arrest lead to differential patterns of cell cycle regulator expression and phosphorylation.

<p>(<b>A</b>) Cells were plated at 30% confluence in 10-cm plates in DMEM containing 10% FBS. After 24 hr, the cells were shifted to CM or blocking conditions for 4 hr, at which time the cells were harvested and the levels of the indicated protein or phosphoprotein was determined by Western blot analysis. The data shown are representative of experiments repeated at least two times. (<b>B</b>) Quantitative analysis of relative protein levels for Western blots shown in (<b>A</b>) using ImageJ software. (<b>C</b>–<b>F</b>) BJ cells were plated and shifted to various blocking conditions for 48 hr as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074157#pone-0074157-g001" target="_blank">Figure 1A</a>. The cells were subsequently released by shifting to CM, and the cells were harvested and lysates collected at indicated time points. The levels of the indicated protein or phosphoprotein were determined by Western blot analysis. The data shown are representative of experiments repeated at least two times. Also shown in the line graphs are the kinetic analyses of relative protein levels normalized to actin and quantitated using ImageJ.</p

Temporal mapping of the metabolic checkpoints from G0.

<p>(<b>A</b>) Schematic representation of the experiment shown in (<b>B</b>). (<b>B</b>) BJ cells were plated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074157#pone-0074157-g001" target="_blank">Figure 1A</a> for 24 hr. Cells were synchronized in G0 by shifting to DMEM+1mM Q lacking GF for 48 hr. The cells were released from G0 by shifting to CM containing DMEM (1mM Q) and 1 µCi/ml [³H]-TdR. Various blocking conditions along with [³H]-TdR were applied at indicated time points. After 36 hr from the release from G0, cells were collected and the incorporated label was determined. This experiment utilized DMEM with reduced Q (1 mM vs. 4 mM) because Q withdrawal following DMEM with high Q did not give strong G1 arrest. Error bars represent the standard error for experiments repeated three times.</p

Growth factor and amino acid deprivation, as well as mTOR inhibition induce G1 cell cycle arrest.

<p>(<b>A</b>) BJ hTERT cells were plated at 20% confluence in DMEM containing 10% FBS for 24 hr at which time they were shifted to complete medium (CM) or various blocking conditions [-GF, -EAA, -Q, +Rapamycin (20 µM)] for 24 or 48 hr. The blocking conditions for Q used DMEM lacking Q; and for EAA, DMEM lacking Leu, Lys, and Arg as described in Material and Methods. The CM contained 10% dialyzed FBS (DFBS) instead of 10% FBS. Cells were labeled with [³H]-TdR for the final 24 hr of treatment, after which the cells were collected and the incorporated label was determined by scintillation counting as described in Materials and Methods. Values were normalized to the cpm for CM, which was given a value of 100%. Total cpm for the CM controls was 60,512 +/- 6529 for the 24 hr time point and 80,427 +/- 3567 for the 48 hr time point. Error bars represent the standard deviation for experiments repeated at least two times. (<b>B</b>) BJ cells were plated and shifted to CM or various blocking conditions for 48 hr as in (<b>A</b>), after which the cells were harvested and analyzed for cell cycle distribution by measuring DNA content/cell as described in Materials and Methods. Error bars represent the standard error from independent experiments repeated four times. (<b>C</b>) To investigate the kinetics for progression into S-phase, BJ cells were plated and shifted to blocking conditions for 48 hr as in (<b>A</b>). Cells were subsequently released by shifting to complete medium, and pulsed with [³H]-TdR at the indicated time points for 1 hr – after which the cells were collected and the incorporated label was determined. Error bars represent the standard error of mean for experiments repeated three times.</p

GF, EAA, Q, and rapamycin mediated G1 cell cycle arrests are distinct and distinguishable.

<p>(<b>A</b>–<b>D</b>) BJ hTERT cells were plated and shifted to various first blocking conditions for 48 hr as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074157#pone-0074157-g001" target="_blank">Figure 1A</a>. The cells were subsequently shifted to CM or different second block conditions containing [³H]-TdR for 24 hr, after which the cells were collected and the incorporated label was determined. Error bars represent the standard error for the experiment repeated at least four times. (<b>E</b>) Schematic model showing relative positions of different metabolic checkpoints relative to R (not drawn to represent precise time scales). G1-pm is post-mitotic phase in G1, G1-ps is pre-S phase of G1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074157#B4" target="_blank">4</a>].</p

Metabolic checkpoints are dysregulated in cancer cells.

<p>MCF7 (<b>A</b>), MDA-MB-231 (<b>B</b>), and Panc-1 (<b>C</b>) cells were plated at 20% confluence in 10-cm plates in DMEM containing 10% FBS. After 24 hr, the cells were shifted to CM or various blocking conditions for 48 hr, at which time the cells were harvested, fixed, stained with propidium iodide, and analyzed for distribution in different phases of cell cycle by measuring DNA content/cell as described in Materials and Methods. Error bars represent the standard error from independent experiments repeated four times. Table with the mean and standard error for the graphs is also shown. (<b>D</b>) Representative flow histograms showing increases in S- and G2/M-phase cell population upon EAA and Q deprivation in MDA-MB-231 and Panc-1 cells.</p