Fig. 3

Phosphorylation of LDHB S162 alters its enzymatic activities. a Diagram of the bi-directional reactions catalyzed by tetrameric LDH, comprising LDHA and LDHB. b In DLD1 cells, the endogenous LDHB was replaced by shRNA-resistant and FLAG-tagged LDHB WT or S162A/D mutants. The expressions of LDHA/B were examined by WB. c FLAG-tagged WT, S162A, S162D of LDHB and LDHA were purified by IP and subjected to measure the bi-directional activities. d His-tagged LDHB WT and S162D were expressed in E. coli. The purified proteins were subjected to measure the bi-directional activities. e WT-Aurora-A or KD-Aurora-A was expressed in the DLD1 cells tested in b, FLAG-tagged proteins were purified by IP and subjected to measure the bi-directional activities. f His-tagged LDHB was incubated with ATP, TPX2(1–25aa) and GST or GST-Aurora-A. Bi-directional activities of LDHB were measured. g The plasmid of NADH/NAD⁺ sensor SoNar was transfected into DLD1 cells used in b. Cells were subjected to live cell imaging. Two channels of emission signals were collected near-simultaneously at 15 s intervals before and after addition of pyruvate (5 mM). The differential interference contrast (DIC) images of the cells (left) and the ratio images of F425/485 were shown. Aurora-A was inhibited by MLN8237 (200 nM, 6 h) before imaging. Scale bar, 10 μm. h Quantitation of NADH/NAD⁺ ratio (presented as F425/485) in g. Arrow indicates the addition of pyruvate at time 0. i NADH/NAD⁺ ratios were measured in empty vector (EV) and Aurora-A overexpressing cells at 10 s intervals. DIC and ratio images of F425/485 were shown. Scale bar, 10 μm. j Quantitation of NADH/NAD⁺ ratio in i. The error bar in panels c, d, e, f represents the standard error of mean(SEM), n = 4 independent experiments in panels c, 3 in panels d, f and 7 in panels e. Source data are provided as a Source Data files. (Student’s t-test *p < 0.05, **p < 0.01, ***p < 0.001, ns not significant).