Fig. 1. SOX9 represses Wnt/β-catenin signaling in mammalian cells.

(A and B) TopFlash reporter luciferase assays in HEK293T cells treated for 16 hours with purified Wnt3a (A) or GSK3 inhibitor CHIR99021 (CHIR) (B) and transfected with a SOX9 expression plasmid. (C) Same as (A) and (B) except Wnt signaling was activated by transfection of N-terminally mutated (S33Y) β-catenin (β-catenin*) and with an increasing dose of SOX9. (D) Same as (C) except an Axin2 enhancer reporter was used instead of TopFlash. (E) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses of SOX9 and two Wnt targets performed in pTRE-SOX9^WT cells. Dox treatment was 13.5 hours. (F) TopFlash reporter assays of several cell lines transfected with β-catenin* with or without SOX9. See Results and Materials and Methods for details about each cell line. Each bar represents the means of biological triplicates ± SD. *P < 0.05; **P < 0.005; ***P < 0.001.