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Fig. 2. I-mfa inhibits endogenous Wnt/ß-catenin signaling in P19 cells. (A) P19 cells, as shown in Fig. 1B,C, were collected for RNA isolation and semi-quantitative RT-PCR analysis as described in the Materials and Methods. (B) I-mfa suppresses LEF-1 reporter activity in P19 cells. Cells were first transfected with control siRNA (si-Ctr) or I-mfa siRNA (si-I-mfa) and two days later with the LEF-1 reporter system. Twenty-four hours later, cells were treated with control-conditioned medium (Ctr CM) or Wnt3a-conditioned medium (Wnt3a CM) for an additional 8 hours before the activity was determined. (C) I-mfa siRNA elevate the expression of the Wnt target gene. Individual clones for different kinds of P19 stable cell line were cultured as a monolayer, and treated with Ctr CM or Wnt3a CM for 6 hours before RNA isolation. Semi-quantitative RT-PCR was performed for cyclin D1 and GAPDH detection. Relative mRNA levels of cyclin D1 were quantified by Labwork 2.0 UVP software and normalized with the amount of GAPDH. (D) I-mfa does not affect Wnt-induced free ß-catenin accumulation. P19 cells were transfected with I-mfa, GSK3ß and Axin1 expression plasmids or LacZ as indicated in the figure. Eighteen hours post-transfection, cells were treated with Ctr CM or Wnt3a CM for a further 6 hours and then collected for cell-fraction preparation and ß-catenin ELISA assay.
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