Figure 2.

(A) Northern blot analysis of WISP-1 RNA in C57MG cells expressing mutant β-catenin or Wnt-1. Total RNA (10 μg) from the cells was loaded onto each lane. The blot was hybridized with a full-length mouse WISP-1 probe. For loading control, the blot was rehybridized with a probe for mouse GAPDH gene. (B) Response of the WISP-1 promoter luciferase reporter (pGL2B-WP, WISP-1 sequence −5882 ∼−42, translation start site as +1) to Wnt-1. C57MG cells were cotransfected with 1 μg of pGK-hygro and 10 μg of pGL2B, pGL2B-WP, or TopFlash. Two days after transfection, hygromycin was added to the medium for selection. Resistant colonies were pooled as stable cell lines. In the coculture experiments, QT6 (solid bars) and QTWnt-1 (open bars) cells were plated on 10-cm plates at a density so that after 48 hr they would be ∼40% confluent. The sample numbers of pGL2B, pGL2B-WP, or TopFlash-transfected C57MG cells were then plated on tissue culture dishes containing either QT6 or QTWnt-1 cells. Luciferase analyses were carried out 48 hr after coculture. (C) WISP-1 promoter response to increasing amounts of β-catenin. The 293 cell were transfected with 0.5 μg, 1.0 μg, or 1.5 μg of empty pCS2/MT vector or different β-catenin mutants together with 0.5 μg of pGL2B-WP. A total of 0.1 μg of CMV–β-gal reporter was cotransfected with each sample to normalize transfection efficiency. Empty pCS2/MT vector was added to transfections to make a total of 2 μg of DNA. Luciferase analysis was carried out 60 hr after transfection. All luciferase measurements are expressed as means ±s.d. of triplicate cultures.