CtBP1 and FANCD2 regulate DKK1 expression. (A) Up- and downregulation of genes involved in Wnt pathways found in HeLa cells depleted of CtBP1/CtBP2 (CtBPi) or FANCD2 (FANCD2i) from microarray data. Data represent fold change in mRNA expression relative to control cells expressing scrambled shRNA. (B) DKK1 expression analysis in HeLa cells transduced with shRNA against CtBP1/CtBP2 (CtBPi) or FANCD2 (FANCD2i). DKK1 mRNA expression was quantified via quantitative PCR (q-PCR) and normalized to expression of the housekeeping gene HPRT1. Data are expressed as the mean fold changes ± SEM relative to control cells expressing scrambled shRNA from 4 different experiments performed in duplicate. (C) DKK1 levels found in HeLa cells depleted of CtBP1 (CtBP1i), FANCD2 (FANCD2i), or both (CtBP1/FANCD2i). DKK1 protein levels in cell culture media were determined using a DKK1 detection ELISA kit and compared with control cells expressing scrambled shRNA (Control). Results are shown as the mean DKK1 concentration ± SEM of 3 independent experiments performed in duplicate, normalized to cell numbers and culture media volumes. (D) Serum samples from 10-month-old wild-type (WT; n = 7), FancA^−/− (n = 10), and FancC^−/− (n = 4) mouse littermates were collected, and Dkk1 plasma levels were measured in duplicates using a Dkk1 detection ELISA kit. (E) Luciferase reporter assays performed in HeLa cells depleted of CtBP1, FANCD2 or CtBP1/FANCD2 following transfection of the pTOPflash TCF/LEF reporter construct or the pFOPflash TCF/LEF mutant construct. Results were compared with control cells expressing scrambled shRNA. Results are expressed as percent TOPflash/FOPflash activity of control cells from 4 different experiments performed in duplicate. * P ≤ .05, **P ≤ .01, *** P ≤ .001.