Journal: eLife

Article Title: Mammalian cell growth dynamics in mitosis

doi: 10.7554/eLife.44700

Figure Lengend Snippet: ( a ) Schematic of growth regulation pathways. Chemical and genetic inhibitors (red), kinases (yellow) and the measured downstream consequences (green) are shown. 1NM-PP1-mediated inhibition of CDK1 is dependent on kinase mutation. ( b ) L1210 cell levels of phosphorylated 4E-BP1 (Thr37/46) in G2 (blue) and mitosis (red) after 2 hr treatment with 250 nM TORIN-1, 1 µM RO-3306 or 50 nM OTSSP167. (n = 5–6 separate cultures). ( c ) Protein synthesis rates of G2 (blue) and mitotic (red) L1210 cells after 2 hr treatment with 1 µM RO-3306 or 50 nM OTSSP167. (n = 6 separate cultures). ( d ) Mass-normalized MAR of control, 1 µM RO-3306 or 30 nM OTSSP167-treated L1210 cells. Solid dark lines indicate the mean and light areas represent ± SEM. Arrows reflect typical time of G2/M transition for each sample. n refers to number of individual cells analyzed. Drug treatments started 1–4 hr prior to mitotic entry and were maintained through the experiment. ( e ) DT40 CDK1as cell protein synthesis rates in G2 (blue) and mitosis (red) after 5 hr treatment with 1NM-PP1. (n = 5–8 separate cultures). ( f ) Mass-normalized MAR of control or 200 nM 1NM-PP1-treated DT40 CDK1as cells. Solid dark lines indicate the mean and light areas represent ± SEM. Arrows reflect typical time of G2/M transition for each sample. n refers to number of individual cells analyzed. Drug treatments started 1–4 hr prior to mitotic entry and were maintained through the experiment. ( g ) Protein synthesis rates of G2 (blue) and mitotic (red) L1210 cells expressing scrambled or 4E-BP1 targeting shRNAs. The cells were treated for 2 hr with 1 µM RO-3306 before sample preparation. (n = 6 separate cultures). ( h ) Protein synthesis rates of G2 (blue) and mitotic (red) L1210 cells after 2 hr treatment with 1 µM RO-3306, 5 hr treatment with 50 µM 4EGI-1 or combined treatment with RO-3306 and 4EGI-1. (n = 6–8 separate cultures). All p-Values were obtained using ANOVA followed by Tukey’s posthoc test.

Article Snippet: The cells were then incubated o/n in 4°C with 4E-BP1 monoclonal antibody (53H11, Cell Signaling Technology, #34470), p-4E-BP1 monoclonal antibody (Thr37/46, 236B4, conjugated to PE, Cell Signaling Technology, #7547S), p-S6RP monoclonal antibody (S235/236, D57.2.2E, conjugated to Alexa 647, Cell Signaling Technology, #4851S) or isotype specific controls (Rabbit mAb IgG, conjugated to PE or Alexa 647, Cell Signaling Technology, #5742S) in PBS solution containing 5% BSA.

Techniques: Inhibition, Mutagenesis, Expressing, Sample Prep

Journal: eLife

Article Title: Mammalian cell growth dynamics in mitosis

doi: 10.7554/eLife.44700

Figure Lengend Snippet: ( a ) L1210 cell levels of phosphorylated 4E-BP1 (Thr37/46) in mitosis and G2. Antibody isotype control is shown as a specificity control. (n = 5–6 separate cultures). p-Values obtained using ANOVA followed by Tukey’s posthoc test. ( b ) Representative image of L1210 cells in interphase (light blue arrow heads) and in early mitosis (yellow arrows) with DNA, α-Tubulin and p-4E-BP1 (Thr37/46) labeling (n = 15 fields of view). Mitotic cells were identified based on DNA condensation and α-Tubulin morphology. DNA and p-4E-BP1 (Thr37/46) labeling within the orange box are shown separately on the right. Scale bars denote 10 µm. ( c ) L1210 cell levels of phosphorylated 4E-BP1 (Thr37/46) in mitotic cells treated with or without Lambda protein phosphatase. (n = 3 separate cultures). p-Values obtained using two-tailed Welch’s t-test. See Materials and methods for experimental details.

Article Snippet: The cells were then incubated o/n in 4°C with 4E-BP1 monoclonal antibody (53H11, Cell Signaling Technology, #34470), p-4E-BP1 monoclonal antibody (Thr37/46, 236B4, conjugated to PE, Cell Signaling Technology, #7547S), p-S6RP monoclonal antibody (S235/236, D57.2.2E, conjugated to Alexa 647, Cell Signaling Technology, #4851S) or isotype specific controls (Rabbit mAb IgG, conjugated to PE or Alexa 647, Cell Signaling Technology, #5742S) in PBS solution containing 5% BSA.

Techniques: Labeling, Two Tailed Test

Journal: eLife

Article Title: Mammalian cell growth dynamics in mitosis

doi: 10.7554/eLife.44700

Figure Lengend Snippet: ( a ) Histogram of 4E-BP1 protein levels analyzed by immunostaining and flow cytometry in L1210 cells stably expressing the indicated shRNAs. ( b ) Quantifications of data in panel ( a ). n = 3 separate cultures. Note that the difference in 4E-BP1 knockdown efficiency between the two 4E-BP1 targeting shRNAs is likely to explain the differences observed between these two cell lines in . ( c ) Proliferation rate of control and 4E-BP1 knockdown cells. Note that the lack of strong proliferation phenotypes likely reflects the optimal growth conditions where 4E-BP1 is maintained phosphorylated and inactivated.

Article Snippet: The cells were then incubated o/n in 4°C with 4E-BP1 monoclonal antibody (53H11, Cell Signaling Technology, #34470), p-4E-BP1 monoclonal antibody (Thr37/46, 236B4, conjugated to PE, Cell Signaling Technology, #7547S), p-S6RP monoclonal antibody (S235/236, D57.2.2E, conjugated to Alexa 647, Cell Signaling Technology, #4851S) or isotype specific controls (Rabbit mAb IgG, conjugated to PE or Alexa 647, Cell Signaling Technology, #5742S) in PBS solution containing 5% BSA.

Techniques: Immunostaining, Flow Cytometry, Stable Transfection, Expressing

Journal: eLife

Article Title: Mammalian cell growth dynamics in mitosis

doi: 10.7554/eLife.44700

Figure Lengend Snippet: ( a ) MAR during indicated stages of cell cycle in control, 1 µM RO-3306, 30 nM OTSSP167 or 100 nM cycloheximide (CHX) treated L1210 cells. The MAR values were normalized to control mean at each stage. n refers to number of individual cells analyzed. Drug treatments started 1–4 hr prior to mitotic entry and were maintained through the experiment. ( b ) MAR during indicated stages of cell cycle in control or 200 nM 1NM-PP1-treated DT40 CDK1as cells. The MAR values were normalized to control mean at each stage. n refers to number of individual cells analyzed. Drug treatment started 1–4 hr prior to mitotic entry and was maintained through the experiment. ( c and d ) MAR of newborn L1210 G1 cells from control and from cells that have undergone mitosis in the presence of 1 µM RO-3306 ( c ) or from cells that have been arrested to mitosis for 4 hr with STLC before releasing to undergo cytokinesis ( d ). Data acquired using serial SMR (see for details). n refers to number of individual cells analyzed. ( e ) Protein synthesis rates of G1 L1210 cells expressing a scrambled or 4E-BP1 targeting shRNA after the cells progressed through mitosis in the presence or absence of 1 µM RO-3306. Two timepoints (3 hr and 8 hr) after G2 release are shown. (n = 4 separate cultures). ( f ) Long-term growth, as measured by cell confluency, in L1210 cells that have been arrested to mitosis for 4 hr with STLC or MG132 before releasing to undergo cytokinesis (n = 5 separate cultures, top), or have undergone mitosis in the presence of 1 µM RO-3306 (n = 6 separate cultures, bottom). Dark colors indicate mean and light areas indicate ± SEM. In ( a ) and ( e ), p-values obtained using ANOVA followed by Tukey’s posthoc test. In ( b–d ), p-values obtained using two-tailed Welch’s t-test. In boxplots, line: median, box: interquartile range, whiskers: 5–95%.

Article Snippet: The cells were then incubated o/n in 4°C with 4E-BP1 monoclonal antibody (53H11, Cell Signaling Technology, #34470), p-4E-BP1 monoclonal antibody (Thr37/46, 236B4, conjugated to PE, Cell Signaling Technology, #7547S), p-S6RP monoclonal antibody (S235/236, D57.2.2E, conjugated to Alexa 647, Cell Signaling Technology, #4851S) or isotype specific controls (Rabbit mAb IgG, conjugated to PE or Alexa 647, Cell Signaling Technology, #5742S) in PBS solution containing 5% BSA.

Techniques: Expressing, shRNA, Two Tailed Test

Journal: eLife

Article Title: Mammalian cell growth dynamics in mitosis

doi: 10.7554/eLife.44700

Figure Lengend Snippet:

Article Snippet: The cells were then incubated o/n in 4°C with 4E-BP1 monoclonal antibody (53H11, Cell Signaling Technology, #34470), p-4E-BP1 monoclonal antibody (Thr37/46, 236B4, conjugated to PE, Cell Signaling Technology, #7547S), p-S6RP monoclonal antibody (S235/236, D57.2.2E, conjugated to Alexa 647, Cell Signaling Technology, #4851S) or isotype specific controls (Rabbit mAb IgG, conjugated to PE or Alexa 647, Cell Signaling Technology, #5742S) in PBS solution containing 5% BSA.

Techniques: Generated, Transfection, Construct, shRNA, Isolation, Activity Assay, Software