Inhibition of Wnt signaling pathway by attenuation of Bmal1 function. A, B) Gene expression analysis by quantitative RT-PCR in 10T1/2 cells (A) and Bmal1−/− MEF cells (B) at d 0–6; n = 3. HET, heterozygote; KO, knockout (homozygote). *P < 0.05, **P < 0.01; Student's t test. C, D) Immunoblot analysis of β-catenin protein level in Cyt fraction and total lysate in WT and Bmal1−/− MEF cells (C) and Bmal1-KD and Bmal1-overexpressing (cDNA) 10T1/2 cells with and without Wnt3a treatment (D). Right panels show quantification of cytosolic to total lysate ratio (n=3). *P < 0.05, **P < 0.01; ##P < 0.01. E) TOPFlash luciferase activity with and without Wnt3a in Bmal1-KD, Bmal1-overexpressing, and control 10T1/2 cells. Values are expressed as TOPFlash reporter activity normalized to Renilla readings after FOPFlash subtraction (n=4). **P < 0.01; ##P < 0.01.