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. Author manuscript; available in PMC: 2020 Jul 18.
Published in final edited form as: Cell Chem Biol. 2019 May 2;26(7):980–990.e8. doi: 10.1016/j.chembiol.2019.03.012

Figure 4 |. mTORC1 and CDK4/6 cooperate to regulate 4E-BP1 phosphorylation and initiation of cap-dependent translation.

Figure 4 |

A, Western blots of 3xFLAG-CDK4 and/or myc-D-cyclin transfected HEK293T cells stimulated and treated with rapamycin or DMSO following serum deprivation. B, Cells were deprived of serum, followed by stimulation with media containing serum and insulin with or without rapamycin and/or palbociclib. C, Colony formation of MCF-7, MDA-MB-468 and MDA-MB-231 cell lines treated with rapamycin and/or palbociclib. D, Quantification of cell density from (c). Signal intensity is normalized to the no treatment control. Error bars indicate standard deviation for 3 biological replicates. E, Western blot of MCF-7, MDA-MB-468 and MDA-MB-231 cell lines treated with rapamycin and/or palbociclib. Vertical bars separate samples run on separate blots or different exposures. F, Cap-dependent dual luciferase assay of cells treated with rapamycin, palbociclib and/or INK128. Normalized Renilla luciferase is shown relative to the no-treatment control for each cell line. Data represented as mean +/− standard deviation (n=4), and are representative of 3 independent replicates. *P<0.05, **P<0.01 ***P<0.001.