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Figure 4 | Breast Cancer Research

Figure 4

From: Autocrine WNT signaling contributes to breast cancer cell proliferation via the canonical WNT pathway and EGFR transactivation

Figure 4

Wnt1 induces rapid phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in human breast cancer cells. (a) Cultures of the indicated cell lines were treated for 20 minutes with Wnt1 conditioned medium (CM) or control CM, and lysates were analyzed by SDS-PAGE followed by immunoblotting for p-ERK1/2 and ERK1/2. (b) T47D cells were treated with control CM or Wnt1 CM, which was previously incubated for 2 hours with 30 μg/mL purified secreted Frizzled-related protein 1 (sFRP1) or phosphate-buffered saline as control. Cell lysates were analyzed by SDS-PAGE/immunoblotting for p-ERK1/2 and p38 as loading control. (c) Stably Wnt1-transfected T47D and SkBr3 cells were treated for 2 hours with sFRP1 CM, control CM, or normal growth medium. Total lysates were analyzed by SDS-PAGE followed by immunoblotting for p-ERK1/2 and ERK1/2. (d) T47D/Wnt1 and SkBr3/Wnt1 cells were seeded at 300,000 cells per well in a six-well plate the day before short interfering RNA (siRNA) transfection with either a LacZ control siRNA or pan-DVL siRNA. The cells were cultured for an additional 48 hours under normal growth conditions and 24 hours in 0.1% fetal calf serum before harvesting. Total lysates were analyzed by SDS-PAGE/immunoblotting for p-ERK1/2, ERK1/2, DVL1, DVL2, and DVL3, or α-Tubulin as a loading control. (e) T47D cultures were treated for the indicated times with 100% and 20% vol/vol Wnt1 CM. Total lysates were analyzed by SDS-PAGE/immunoblotting for p-ERK1/2 and ERK1/2 (upper panel). ERK activation was quantified after normalization of signal intensity of p-ERK1/2 to total ERK1/2 using the ImageQuant software. ERK activity peaks at between 30 minutes and 1 hour (lower panel). (f) Wnt1-mediated effects are independent of β-catenin. T47D cells were infected with a retrovirus carrying an expression cassette for a short-hairpin RNA targeting β-catenin. Two independent shβ-catenin clones and a control pool infected with a short hairpin against bacterial LacZ were treated with control CM or Wnt1 CM for 20 minutes or 10 ng/mL EGF for 5 minutes. Total lysates were analyzed by SDS-PAGE/immunoblotting for p-ERK1/2, ERK1/2, and β-catenin. DVL, Dishevelled.

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