Figure 2. CTDSP2 expression is regulated by FOXO proteins.

(A) CTDSP2 mRNA was measured by qPCR in DL23 cells after FOXO3 was activated by 4OHT for the indicated times or (B) for 8 h in the presence of cycloheximide (CHX). (C) U2OS cells were treated for 16 h with LY294002 or Akti to inhibit PI3K and PKB/Akt and thereby activate endogenous FOXO proteins. CTDSP2 mRNA was measured by qPCR. Either treatment induces CTDSP2 expression significantly. (D) ChIP–qPCR-determined percentage input of the CTDSP2 promoter precipitated by endogenous FOXO3 (black bars) or non-specific IgG (white bars). Nuclear localization of endogenous FOXO3 was induced using LY294002 and precipitated using anti-FOXO3 antibody. (E) U2OS cells were transfected as indicated in the Materials and methods section. FOXO3.A3 induces luciferase expression driven by a 479 bp fragment overlapping the 5′-UTR of human CTDSP2 (white bars), which is decreased if the FOXO-binding sites are mutated (black bars). WT, wild-type. Results are means±S.D. of three biological replicates or representative examples in the case of ChIP. Student's t tests: *P<0.05, **P<0.005; indication absent, P>0.05. NT, not treated.