Extended Data Fig. 1: Glycolysis is required for cell cycle entry.
From: Transient APC/C inactivation by mTOR boosts glycolysis during cell cycle entry
a, Immunoblot analysis of the indicated proteins from either cycling or mitogen starved MCF-10A cells. Representative blot of n = 3 independent experiments. b, Schematic of the cyclin E/A-CDK1/2 (CDK2) biosensor. A fragment of human DNA helicase B (amino acids 994-1087) fused to mVenus under the control of the constitutive promoter EF1α. c, MCF-10A cells were starved for 48 h to induce quiescence followed by mitogen stimulation with DMSO or 2-deoxy glucose (2-DG). Percent of cells turning CDK2 on were quantified. Data are mean ± SD from n = 3 independent experiments. P-values were calculated using a one-way ANOVA. ns, Not significant. d, Experiment to test whether OXPHOS is needed to start the cell cycle (left). CDK2 activity traces of single cells for the indicated treatments since the time of mitogen addition (right). MCF-10A cells were starved for 48 h to induce quiescence followed by mitogen stimulation with or without the addition of oligomycin (Oligo) at the indicated concentration. Green traces depict cells that entered the cell cycle. Grey traces depict cells that remain quiescent and failed to enter cell cycle. N = 200 cells per condition. e, Experiment to test whether glucose is needed to start the cell cycle. CDK2 activity traces of single cells for the indicated treatments since the time of mitogen or EGF addition. MCF-10A cells were starved for 48 h to induce quiescence by switching the growth media to growth media minus growth factors (GM-GFS). At time zero, the media was switched to either full growth media, GM-GFS supplemented with EGF and with and without glucose (Glu). Green traces depict cells that entered the cell cycle. Grey traces depict cells that remain quiescent and failed to enter cell cycle. N = 200 cells per condition. f, Experiment to test whether glucose transport is needed to start the cell cycle. CDK2 activity traces of single cells for the indicated treatments since the time of mitogen addition. MCF-10A cells were starved for 48 h to induce quiescence followed by mitogen stimulation with or without the addition of 30 μM DRB18, a pan GLUT inhibitor. Green traces depict cells that entered the cell cycle. Grey traces depict cells that remain quiescent and failed to enter cell cycle. N = 200 cells per condition. g-i, MCF-10A (g), retinal pigment epithelial cells (RPE-1) (h), or mouse embryonic fibroblasts (MEF) (i) were grown in serum free medium for 48 h to induce quiescence. At time 0 h, cells were stimulated with growth medium containing 5% fetal bovine serum with either glucose, galactose, or neither. After 16 h, cells were harvested and cell lysates were subjected to immunoblot analysis for phosphorylated Rb, a marker for cell cycle entry. Representative blot of n = 2 independent experiments.
