Fig. 5.

PKM2 O-GlcNAcylation is crucial for the Warburg effect and tumor growth. (A) O-GlcNAcylation of PKM2^WT enhances the mRNA level of Glut1 (Left) and LDHA (Right). Error bars, SEM; n = 4 biological replicas. Student’s t test: ***P < 0.001; ns, nonsignificant. (B) Protein levels of Glut1 and LDHA were induced by PKM2 O-GlcNAcylation. (C) Establishment of PKM2 rescue cell lines. MCF-7 cells depleted for endogenous PKM2 were stably reexpressed with exogenous PKM2^WT or PKM2^T405A/S406A. (D) Proliferation curves of PKM2^WT and PKM2^T405A/S406A rescue cell lines. PKM2^WT and PKM2^T405A/S406A rescue cells were seeded at the same number in each well. Cell numbers were counted every 24 h. Error bars, SEM. Student’s t test: *P < 0.05; ***P < 0.001; ns, nonsignificant. (E–G) Tumor formation in nude mice. PKM2^WT and PKM2^T405A/S406A rescue cells were, respectively, injected into athymic nude mice. The xenograft tumors were sampled and photographed after 24 d. Images of mice bearing tumor are as in E. Tumor volumes were measured at the indicated time points as in F. According to length (l), width (w), and height (h), volumes were calculated based on the equation v = lwhπ/6. The quantification of the average mass of xenograft tumors was as in G. Mean is shown, n = 9. Student’s t test: *P < 0.05; **P < 0.01; ***P < 0.001; ns, nonsignificant. (H) Immunohistology analysis for cell proliferation. Nine pairs of mice injected with PKM2^WT or PKM2^T405A/S406A rescue cells were costained with hematoxylin and eosin (H&E) and Ki-67. (H, Left) Representative images depicting tumor tissues. (Scale bars, 50 μm.) (H, Right) Quantification of Ki-67 staining. Error bars, SEM; n = 15 (five fields from each of the three analyzed mice). Student’s t test: **P < 0.01.