Skip to main content
. 2006 Oct 30;27(2):466–480. doi: 10.1128/MCB.01539-06

FIG. 8.

FIG. 8.

Wnt-1 expression links PR transcriptional activity to EGFR transactivation and sustained MAPK signaling. (A) The time course of R5020-treated (+) T47D-YB cells demonstrates specific upregulation of Wnt-1 mRNA by RT-PCR. The results are representative of four independent experiments. (B) Duplicate cultures of untreated T47D-YB parental (YB) cells or cells stably expressing Neg shRNA control (N2) or Wnt-1 shRNA (Wnt-1) were treated for 18 h with R5020, and knockdown of Wnt-1 mRNA expression was measured by RT-PCR. (C) EGFR from T47D-YB cells stably expressing control shRNA (Neg) or Wnt-1 shRNA (Wnt-1) was immunoprecipitated using specific antibodies or control IgG and Western blotted using antibodies specific for phospho-Y1173 (p-Y1173) (activated) EGFR and total (t) EGFR. Lysates indicate 10% IP input. Normalization of phospho-Y1173 to total EGFR in immunoprecipitates, as measured by densitometry, indicated EGFR activity was increased 1.9-fold in R5020-treated control (Neg) cells, whereas cells expressing Wnt-1 shRNA exhibited no increase in EGFR activity (0.9-fold compared to the EtOH signal); densitometry of multiple exposures yielded similar results. The results were confirmed in two independent experiments. (D) Duplicate cultures of wt T47D-YB (YB) cells or cells stably expressing either control (Neg) or Wnt-1 shRNA (Wnt-1) were treated without (−) or with (+) R5020 for 18 h, and whole-cell lysates were Western blotted for cyclin D1, EGFR, and phospho- and total Erk1/2 MAPKs. Duplicate measurments of active Erk2 MAPK and cyclin D1 protein bands were quantified by densitometry and are represented as bars in the lower graphs (± standard deviations). (E) Triplicate cultures of T47D-YB (YB parent) cells or cells stably expressing either control shRNA (Neg) or Wnt-1 shRNA (Wnt-1) were plated in soft agar as described in Materials and Methods and treated without (EtOH) or with (black bars) R5020 for 18 h, and soft-agar colonies were counted after 3 weeks. The average colony numbers are presented as n-fold over the EtOH control for the T47D-YB parent, shRNA Neg, or Wnt-1 (± standard errors of the mean). The asterisks denote statistical significance (P < 0.01) determined by an unpaired Student's t test. The experiments were repeated twice with similar results.