Journal: bioRxiv

Article Title: Cell size sensing in animal cells coordinates anabolic growth rates with cell cycle progression to maintain uniformity of cell size

doi: 10.1101/123851

Figure Lengend Snippet: (A-D) Cells were treated with varying doses of drugs inhibiting cell cycle progression (circles) or protein production (squares). (A) The fold change in cell size (after 44 hrs in drug) vs. fold change in growth rate, relative to untreated cells, is plotted for each condition. Dotted line denotes proportional relationship expected if there is no coordination between growth and cell cycle progression. (B) The fold change in cell size (after 44 hrs in drug) vs. fold change in cell cycle length, relative to untreated cells, is plotted for each condition. (C) Mean growth rate vs. mean cell cycle length are plotted for cells in each condition, calculated from measured increases in bulk protein and number of cells over the course of a 68-hour incubation. Black lines mark iso-cell-size contours. Region between lines spans a 25% shift in cell size. (D) Cells were transfected with plasmids encoding wild-type or degradation-resistant cyclin E under control of a constitutive promoter (diamonds). Mean growth rate vs. mean cell cycle length are plotted for cells in each condition, overlaid on the plot shown in (C). Calculation of the Pearson correlation coefficient between log(growth rate) and log(cell cycle length), using the data shown in (D), yields r = -0.76, p = 2.1x10 −7 , by two-tailed Student’s t-test, indicating a significant inverse correlation between growth rate and cell cycle length. (E,F) Mean growth rate vs. mean cell cycle length for cells treated with drugs inhibiting cell cycle progression (E) or protein production (F), calculated for three time intervals during drug treatment: 0-14 hrs, 14-44 hrs, 44-68 hrs. Lines connect time-points in sequential order, with arrowheads pointing to latest time-point. Data is shown for 25 uM BN82002 (E, green), 1.5 uM Cdk2 Inhibitor III (E, red), 0.06 uM Cycloheximide (F, blue), 10 nM Torin (F, orange), and 7 uM Rapamycin (F, purple). For (A-F), each treatment was done in duplicate, with several thousand cells in each sample.

Article Snippet: Cyclin E-pBI-EGFP was a gift from Bert Vogelstein (Addgene plasmid # 16654)( Rajagopalan, H. et al.

Techniques: Incubation, Transfection, Control, Two Tailed Test

Journal:

Article Title: DNA Damage-Induced G 1 Arrest in Hematopoietic Cells Is Overridden following Phosphatidylinositol 3-Kinase-Dependent Activation of Cyclin-Dependent Kinase 2

doi: 10.1128/MCB.21.18.6113-6121.2001

Figure Lengend Snippet: Irradiated 32D cells arrested in G1 have inactive cyclin E-Cdk2 complexes but retain active Cdk4 and Cdk7. Cell lysates from the 32D-EpoR[H] cultures represented in Fig. ​Fig.1B1B were immunoprecipitated (I.P.) with antibodies to Cdk4, Cdk2, cyclin E, or Cdk7, and in vitro kinase assays were performed (Activity). Immunoprecipitated proteins were Western blotted with Cdk4 antibodies (Cdk4 IP), total and phosphorylation site-specific Cdk2 antibodies (Cdk2 and cyclin E IPs), or Mat1 antibodies (I.P. Cdk7).

Article Snippet: Specific proteins were precipitated with 2 μg of antiserum/ml specific for Cdk4 (Santa Cruz C-22), cyclin E (Santa Cruz M-20), or Cdk7 (Santa Cruz M-19) and were used in kinase reactions or for Western blotting.

Techniques: Irradiation, Immunoprecipitation, In Vitro, Activity Assay, Western Blot

Journal:

Article Title: DNA Damage-Induced G 1 Arrest in Hematopoietic Cells Is Overridden following Phosphatidylinositol 3-Kinase-Dependent Activation of Cyclin-Dependent Kinase 2

doi: 10.1128/MCB.21.18.6113-6121.2001

Figure Lengend Snippet: Inhibition of PI 3-kinase prevents cytokine treatment from overriding DNA damage-induced inactivation of cyclin E-Cdk2 and expression of p27. (A) 32D-EpoR[wt] cells were synchronized in G0/G1 in the absence of growth factor (No factor). Synchronized cell cultures were supplemented with LY294002 (10 μM) or vehicle control (dimethyl sulfoxide [DMSO]), plus Epo or IL-3. Parallel cultures were then left untreated or exposed to γ irradiation (+ 4 Gy). All cultures were incubated for an additional 24 h prior to analysis by flow cytometry. Values represent the percentage of cells in S phase. (B) Cell lysates prepared from the cultures represented in panel A were immunoprecipitated with Cdk4 or Cdk2 antibodies, and in vitro kinase assays were performed. Alternatively, total cell lysates were Western blotted with antibodies specific for Cdk2 or p27.

Article Snippet: Specific proteins were precipitated with 2 μg of antiserum/ml specific for Cdk4 (Santa Cruz C-22), cyclin E (Santa Cruz M-20), or Cdk7 (Santa Cruz M-19) and were used in kinase reactions or for Western blotting.

Techniques: Inhibition, Expressing, Irradiation, Incubation, Flow Cytometry, Immunoprecipitation, In Vitro, Western Blot