Figure 4.

Stable Sh3gl2 silencing attenuates EGFR internalization and enhances receptor activation. (A) Control and Sh3gl2-silenced RT4 cells were seeded on coverslips, serum-depleted for 24 hours, and treated without or with 10 nM EGF for 10 minutes at 37°C. Localization of the EGFR was visualized by indirect immunofluorescence staining. Nuclei are counterstained with 4′, 6-diamidino-2-phenylindole (DAPI). Data are representative of three independent trials. Original magnification, x63. (B) The extent of EGFR internalization under the conditions indicated in A was quantified and plotted as the percentage of cells showing EGFR that was localized predominantly on the membrane (Memb), predominantly in the cytoplasm (Cyto), or present both in the cytoplasm and on the membrane (Cyto + Memb). (C) Serum-depleted control and Sh3gl2-silenced RT4 cells were treated with 10 nM EGF for 10 minutes at 37°C, and whole-cell lysates were prepared for evaluation of receptor phosphorylation. Increased EGFR phosphorylation was evident in all four clones in which Sh3gl2 was silenced compared to nontargeted control cells. Data are representative of at least four independent experiments. (D) Control and Sh3gl2-silenced RT4 cells were treated with vehicle (DMSO) or 0.3 µM lapatinib for 5 days and cell number determined by biomass assay. Data represent absorbance on day 5 expressed as a percentage of absorbance on day 0 and are the means ± SD of four values. *, shCtrl C2 DMSO versus sh184 C9 DMSO or sh2103 C8 DMSO; **, shCtrl C2 lapatinib versus sh184 C9 lapatinib or sh2103 C8 lapatinib. Data are representative of at least two independent experiments.