Figure 8. Effect of β-hydroxybutyrate (HB) on FoxO1-dependent gene expression after FoxO1 knockdown. Western blot analysis was used to assess protein levels in FoxO1 siRNA-treated HEK293T cells. (A) FoxO1, PGC-1α, and NF-κB protein levels in cells pretreated for 3 h with HB in the absence or presence of FoxO1 siRNA-transfected cells (200 MOI) for 1 day. (B) HEK293T cells were transiently transfected with a catalase-containing plasmid linked to the luciferase gene, pre-incubated with FoxO1-siRNA (100 MOI) for 24 h, and then treated with HB for 4 h. Results are presented in relative luminescence units (RLU). Results were obtained using one-factor ANOVA: $$$p<0.001 vs. pcDNA transduced cells; ###p<0.001 vs. catalase-luciferase transduced cells; **p<0.01, ***p<0.001 vs. FoxO1-siRNA with catalase-luciferase transduced cells. (C) HEK293T cells were pretreated with or without 0.5 mM HB for 3 h and then treated with FoxO1-siRNA (200 MOI) for 24 h. Cells were immunostained using rabbit anti-PGC-1α antibody followed by IgG conjugated with fluorescein isothiocyanate (green). Bar = 50 µm. (D) A possible mechanism underlying the effect of HB regulate NF-κB through interaction of FoxO1 and PGC-1α in aging.