Fig. 6

Smyca coordinates with TGF-β and c-Myc pathways for stimulating glycolysis and preventing growth inhibition. (A, B) RIP assays for the enrichment of Smyca in Smad3, Smad4 or c-Myc immunoprecipitates derived from MCF7 cells stably expressing Smyca and treated with or without 5 µM SB431542 for 24 h (A), or MCF7 cells stably expressing Smyca and transfected with c-Myc siRNA (B). (C, D) Cell proliferation (C) and qRT-PCR analysis (D) of MCF7 cells stably expressing Smyca, transfected with c-Myc siRNA, and/or treated with 5 µM SB431542 for 24 h. Validation of c-Myc knockdown efficiency is shown on the left panel in (C). (E, F) Cell proliferation (E) and qRT-PCR analysis (F) of MCF7 cells transfected with indicated Smyca constructs. The expression levels of Smyca and mutants are shown on the left panel in (E). (G, H) Glucose consumption (G) and lactate formation (H) in BT-549 cells transfected with Smyca and treated with 10 µM SB431542 and/or 150 µM 10058-F4 for 24 h (G) or 48 h (H). Validation of Smyca overexpression is shown on the left panel in (G). (I, J) Glycolysis stress profile (I) and glycolysis and glycolytic reserve rates (J) were measured using MDA-MB-231 cells transfected and treated as in (G). Data in all panels are mean ± SD, n = 3. P values are determined by unpaired t test (A, B), one-way (C, D, E, F) or two-way (G, H, J) ANOVA with Tukey’s post hoc test, *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant