FIGURE 4.

SOX9 enhances Wnt3A stimulation of Wnt/β-catenin activity. A and D, MCF10A-DCIS (retroviral transduced) or HCC1937 (lentiviral transduced) cells stably expressing control or SOX9-targeted shRNA were transfected with Topflash-firefly-luc together with CMV-Renilla-luc reporters for 72 h. The cells were treated with mouse Wnt3A during the last 8 h. The activity was presented as relative light unit (RLU) with firefly corrected for Renilla luciferase activity. B and E, MCF10A-DCIS or HCC1937 cells stably expressing control or SOX9-targeted shRNA were treated with mouse Wnt3A for 8 h, and the cell lysates were immunoblotted for SOX9, active (Act-) or total β-catenin and Axin2. C and F, cells were similarly treated as described in B and E. Total RNA was collected, and Axin2 mRNA was measured by quantitative real time RT-PCR. RC, relative change compared with the level in untreated control cell. G, MCF10A-DCIS cells expressing control or SOX9-specific shRNA (shSOX9) were transfected with either mock (pcDNA3) or LRP6 expression vectors (LRP6-pCS2) for 48 h. Carrier or Wnt3A (40 ng/ml) were added during the last 8 h. The LRP6 protein levels were measured by immunoblotting (bottom panels), and Axin2 mRNA levels were measured by quantitative real time RT-PCR. Statistical analysis was performed with Student's t test (n = 3). Error bars, S.E.