Extended Data Fig. 8: Transient accumulation of PFKFB3 is dependent on Cdh1-T129 phosphorylation by mTOR.
From: Transient APC/C inactivation by mTOR boosts glycolysis during cell cycle entry
a, MCF-10A cells infected with pTeton-Cdh1-T129A were mitogen starved for 48 h to induce quiescence. Cells were then treated with increasing concentration of doxycycline for 8 h to induce the expression of HA-Cdh1-T129A. Representative blot of n = 3 independent experiments. b, MCF-10A cells infected with either empty vector or pTeton-Cdh1-T129A were starved for 48 h to induce quiescence followed by mitogen stimulation with 2.5 μM Doxycycline treatment. Cells were collected at the indicated time points. Whole cell lysates were resolved on SDS-PAGE and probed for the indicated proteins. Representative blot of n = 3 independent experiments. c, Quantification of PFKFB3 relative expression in presence of doxycycline inducible Cdh1-T129A, from (b). Data are mean ± SD from n = 3 independent experiments. P-values were calculated using a one-way ANOVA. d, MCF-10A cells infected with either empty vector or pTeton-Cdh1-T129A were starved for 48 h to induce quiescence followed by mitogen stimulation with doxycycline treatment. Cells were collected at the indicated time points. Whole cell lysates were resolved on SDS-PAGE and probed for the indicated proteins. Representative blot of n = 4 independent experiments. e, Quantification of relative PFKFB3 expression from (d). Data is from n = 4 independent experiments. P-values were calculated using a one-way ANOVA. f, MCF-10A cells infected with either empty vector or pTeton-Cdh1-T129A were starved for 48 h to induce quiescence followed by mitogen stimulation with doxycycline treatment. Cells were collected at the indicated time points. Whole cell lysates were resolved on SDS-PAGE and probed for the indicated proteins. Representative blot of n = 4 independent experiments. g, Quantification of relative PFKFB3 expression from (f). Data are mean ± SD from n = 4 independent experiments. P-values were calculated using a one-way ANOVA with Sidak’s multiple comparison test. ns, Not significant. h, MCF-10A cells transfected with vectors to express the indicated protein. WT-PFKFB3, wild-type PFKFB3; PFKFB3KEN mut, KEN box mutant PFKFB3, with and without WT-Cdh1. Whole cell lysates were resolved on SDS-PAGE and probed for the indicated proteins. i, Quiescent MCF-10A cells were transfected with either Cdh1-T129A, PFKFB3KEN mut, or both and stimulated with mitogens for 4 h. Whole cell lysates were resolved on SDS-PAGE and probed for the indicated proteins. j, Quiescent MCF-10A cells transfected with either empty vector or Cdh1-T129A were treated with either DMSO or mitogens for 4 h. Data is mean fold change ± SD of PFKFB3 mRNA from n = 3 independent experiments. P-values were calculated using a one-way ANOVA. ns, Not significant. k, Quiescent MCF-10A cells transfected with either empty vector, Cdh1-T129A, or Cdh1-T129D were treated with either DMSO or mitogens for the indicated time points. Whole cell lysates were probed for the indicated proteins. Representative blot from n = 3 independent experiments.l, Quantification of (i). Data are mean ± SD from n = 3 independent experiments. P-values were calculated using a one-way ANOVA. ns, Not significant.
