Extended Data Fig. 7: Persistent lactate elevation drives mitotic slippage through aberrant APC/C activation.
From: Lactate regulates cell cycle by remodelling the anaphase promoting complex
(a) Treatment of cells with an inhibitor of microtubule assembly nocodazole (sync.) for 14 h to arrest in pro-metaphase leads to accumulation of cyclin B1. Exposure of arrested WT cells to lactate for the last 12 h of nocodazole exposure stimulates depletion of cyclin B1 in the presence of nocodazole. (b) Treatment of HCT116 or HeLa S3 cells with an inhibitor of microtubule assembly nocodazole (sync.) for 14 h to arrest in pro-metaphase leads to accumulation of cyclin B1. Co-exposure of arrested WT cells to 1 µM oligomycin A stimulates depletion of cyclin B1 in the presence of nocodazole. Arrested APC4 K772/798R cells treated with oligomycin A exhibit no oligomycin A dependent effect. (c) Treatment of HCT116 or HeLa S3 cells with an inhibitor of microtubule assembly nocodazole (sync.) for 14 h to arrest in pro-metaphase leads to accumulation of cyclin B1. Co-exposure of arrested WT cells to 5 mM αCHCA stimulates depletion of cyclin B1 in the presence of nocodazole. Arrested APC4 K772/798R cells treated with αCHCA exhibit no αCHCA dependent effect. (d,e) Treatment of (d) WT cells and (e) APC4 K772/798R with an inhibitor of microtubule assembly nocodazole for 14 h to arrest in pro-metaphase. Rate of mitotic progression was determined by timelapse microscopy following exposure of cells to lactate for the last 12 h of nocodazole incubation. Analysis represents time in mitotic arrest beginning with nuclear envelope breakdown (NEBD) to DNA decondensation. n = 30 individual cell replicates. (two-tailed Student’s t-test for pairwise comparisons in d). (f) Treatment of cells with an inhibitor of microtubule assembly nocodazole for 14 h to arrest in pro-metaphase. Quantification of WT cells exhibiting chromosome mis-segregation following treatment with lactate for the last 12 h of nocodazole exposure. n = 30 individual cell replicates. (g) Treatment of cells with an inhibitor of microtubule assembly nocodazole for 14 h to arrest in pro-metaphase. Quantification of APC4 K772/798R cells exhibiting chromosome mis-segregation following treatment with lactate for the last 12 h of nocodazole exposure. n = 30 individual cell replicates. Data are mean ± s.e.m.
