Fig 1: CDK1 drives mitotic growth through 4E-BP1 and cap-dependent protein synthesis.(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.