Fig. 5

Phosphorylation of LDHB S162 promotes glycolysis. a In DLD1 cells, LDHB was knocked down by sh RNA. Seahorse assays were performed to evaluate the glycolytic conditions. ECAR over time (left panel) and ECAR in different stages of the measurement (right panel) were shown. b The expression of LDHA and LDHB in cells used in a were examined by WB. c In DLD1 cells, endogenous LDHB was knocked down, then shRNA-resistant WT LDHB, LDHB S162A/D, and LDHA were expressed. Seahorse assays were performed in these cells to evaluate the glycolytic flux. ECAR over time (left panel) and ECAR in different stages of the measurement (right panel) were shown. d The expression of LDHA/B in cells used in c were examined by WB. e In DLD1 cells used in c, Aurora-A was inhibited by MLN8237 (200 nM, 4 h). Seahorse assays were conducted. ECAR over time (left panel) and ECAR in different stages of the measurement (right panel) were shown. f The expression of LDHA/B and the activity of Aurora-A kinase in cells used in e were examined by WB. g The glycolytic tracing assay was performed with ¹³C-labeled glucose in cells used in c. The relative abundance of the ¹³C-labeled glycolytic metabolites: Glucose-6-phosphate (G6P), phosphoenolpyruvate (PEP), lactate and ribose-5-phosphate (R5P) were shown. h The glycolytic flux assay was performed with ¹³C-labeled glucose in cells used in e. MLN8237 (200 nM, 24 h) was used to inhibit Aurora-A activity. The relative abundance of the ¹³C-labeled glycolytic metabolites were shown. The error bar in panels a, c, e, g, h represents the standard deviation (SD), n = 3 biologically independent samples. 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).