FIGURE 4.

Increased transcript expression of the Pcdhβ gene cluster is caused by impaired her-2-mediated downstream signaling. A, wild-type tumor cells were grown for 2 days with or without MEK inhibitor PD98059 (PD; 10 mol/liter), PI3K inhibitor wortmannin (WM; 1 mol/liter), or both inhibitors. Cells were then collected, and total RNA was isolated and used for detection of transcript levels of Pcdhβ. B, GnT-V knock-out tumor cells were grown in serum-free medium for 2 days and stimulated with EGF (100 ng/ml), neuregulin (NRG) (50 ng/ml), or serum-containing medium for 2 days. Cells were collected for detection of phospho-PKB (p-PKB) and phospho-ERK (p-ERK) using immunoblot (left panel) and transcripts of the Pcdhβ cluster (right panel). After wild-type her-2 cells were treated with control (scrambled) and her-2/neu siRNA oligonucleotides (50 nm) for 48 h, respectively, cells were collected and subjected to detection of transcripts of her-2/neu using qRT-PCR (C); her-2/neu, phospho-PKB (p-PKB), and phospho-ERK (p-ERK) using immunoblot (D); and transcripts of the Pcdhβ gene cluster (E). For each transcript, the values are normalized to control (Gapdh or Rpl4) and expressed as means with error bars indicating ±1 S.D. of three independent experiments. *, Student's t test, p < 0.05; **, p < 0.01.