Extended Data Fig. 5: Timing and effects of lactate accumulation on cell cycle and proliferation.
From: Lactate regulates cell cycle by remodelling the anaphase promoting complex
(a) Schematic of metabolic flux to anabolic precursors tied to lactate production, pathways predicted to increase flux in response to growth demand in G1/S/G2 phase. (b) Outline of experiment to monitor relationship between lactate abundance and cell cycle phases in proliferative cells. Double thymidine block and release synchronized cells at the G1/S interface as described in Methods. (c) Parallel tracking of securin and cyclin B1 abundance (top), cell cycle phase (middle), and intracellular lactate abundance (bottom) in WT and APC4 K772/798R HCT116 cells. n = 6 cell replicates; 12 h APC4 K772/798R n = 5; lactate abundance n = 6; 6 h WT n = 5. Representative blot from three experiments shown. (d) MS quantification of proteins involved in lactate production and lactate abundance in asynchronous HCT116 cells and 0h, 6h, and 12h post-DTB release. n = 4 cell replicates. (e) MS quantification of lysine lactylation in asynchronous HCT116 cells and 0h, 6h, and 12h post-DTB release. n = 4 cell replicates. (f) Abundance of PDHe1α and PDHe1α phosphorylation on residue Ser293 tracked during cell cycle following DTB release. as = asynchronous. Representative blot from three experiments shown. (g) CD8+ T cells stimulated with IL-2 and anti-CD28 for 24 h with cell cycle and intracellular lactate abundance monitored. Lactate abundance n = 4; cell cycle n = 6 cell replicates. (h) Model for lactate accumulation mediating timed remodeling of APC/C to facilitate mitotic progression. (i) ΔCt quantification of bacterial LMO mRNA expression in HCT116 cells relative to β-actin. n = 3 cell replicates. (j) Cells expressing LMO, or treatment of cells with 25 mM DCA, lowers peak intracellular lactate abundance achieved upon mitotic entry. n = 6 cell replicates. (k) Negative correlation between intracellular lactate abundance and doubling time in 707 human proliferative cancers. See Methods for details. (l) Proliferation rate of WT and APC4 K772/798R HeLa S3 and HCT116 cells. Cells lacking APC4 SUMOylation sites exhibit a significantly lower proliferation rate. n = 4 cell replicates. (m) Exposure of asynchronous HCT116 cells to 1% O2 for 24 h lowers levels of securin and cyclin B1. Representative blot from three experiments shown. (n) Proliferation rate of WT, APC4 K772/798R, and LMO-expressing HCT116 cells cultured at 1% O2. n = 5 cell replicates. (two-tailed Student’s t-test for pairwise comparisons in c,j, one-way ANOVA for multiple comparisons in g, two-sided Pearson R in k, two-way ANOVA for multiple comparisons involving two independent variables in l,n). ANOVA analyses were subjected to Bonferroni’s post hoc test.
