Figure 4

Ketone bodies reduce c-Myc expression and its recruitment to glycolytic gene promoters. Recruitment of c-Myc onto GLUT1 (A) and LDHA (B) promoters in S2-013 cells under treatment with 20 mM NaHB, LiAcAc, or control was confirmed by performing ChIP using anti-c-Myc Ab and IgG control, followed by qRT-PCR analysis. Relative c-Myc mRNA levels in Capan1 (C) and S2-013 (D) cells treated with 10 and 20 mM NaHB, LiAcAc, or control for 24 h. Total RNA was isolated and relative mRNA level of c-Myc was determined by qRT-PCR. β-Actin was utilized as an internal control. Capan1 (E) and S2-013 (F) cells were treated with indicated doses of ketone bodies for 48 h, and c-Myc protein level was determined by immunoblotting the whole cell lysates. HSP90 was used as an internal control. (G) c-Myc-promoter-firefly luciferase reporter and Renilla luciferase reporter plasmids were transiently transfected into S2-013 cells. After 16 h of transfection, cells were treated with solvent control or ketone bodies for 24 h. Normalized firefly to Renilla luciferase activity ratio is plotted in the bar chart. Values represented are mean ± SEM. *P < 0.05; **P < 0.01.