Figure 1

Identification of perhexiline as a novel agent that promotes HER3 internalization and degradation. (A) Perhexiline promotes HER3ΔNLS2-YFP internalization. As a control to HER3ΔNLS2-YFP cellular distribution, endogenous HER3 expression in MDA-MB-468 cells was detected by immunofluorescence staining (i). U2OS cells stably expressing HER3ΔNLS2-YFP were used to screen for compounds that promote HER3 internalization. Representative confocal images shown in panels (ii to v) were taken from cells expressing HER3ΔNLS2-YFP that were treated with DMSO, 10 μM perhexiline for 30 minutes and 4 hours, and 20 μM CPT-1 inhibitor etomoxir for 4 hours, respectively. Arrows indicate intracellularly localized puncta of internalized HER3ΔNLS2-YFP receptors. (B) Structure of perhexiline maleate. (C) Perhexiline induces dose-dependent downregulation of endogenous HER3 receptors. Cell lysates prepared from SK-BR-3 cells treated with different concentrations of perhexiline for 8 hours were analyzed for the expression of HER3, HER2, and EGFR. β-actin was used as a loading control. (D) Quantification of HER3, HER2, EGFR protein expression following perhexiline treatment. Western blots shown in (C) were quantified by normalizing to β-actin. (E) Time course of perhexiline-induced downregulation of endogenous HER3 in SK-BR-3 cells. Cells treated with 10 μM perhexiline for the indicated time were analyzed for endogenous HER3, HER2, and EGFR expression. (F) Quantification of HER3, HER2, EGFR protein expression following perhexiline treatment. Western blots shown in (E) were quantified by normalizing to β-actin. CPT-1, mitochondrial carnitine palmitoyltransferase-1; DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; HER3, human epidermal growth factor receptor 3.