Figure 2

Perhexiline promotes cell surface-expressed Flag-HER3ΔNLS2 receptor internalization and the ubiquitination-mediated degradation of HER3 receptors through lysosome and proteasome. (A) Cell surface-expressed Flag-HER3ΔNLS2 receptors are internalized upon perhexiline treatment. HEK293 cells transfected with Flag-HER3ΔNLS2 were incubated with the anti-FLAG antibody on ice for 30 minutes (i) and subsequently incubated at 37°C for 1 hour untreated (ii) or in the presence of DMSO or perhexiline (10 μM) (iii, iv). Cells were fixed and localization patterns of Flag-HER3ΔNLS2 were visualized using confocal microscopy. Internalized Flag-HER3ΔNLS2 vesicles were shown as indicated by arrowheads in DMSO-treated cells whereas arrows indicate large puncta of internalized receptors in perhexiline-treated cells. (B) Perhexiline induces HER3 ubiquitination. MDA-MB-468 cells were treated with perhexiline (10 μM) for the indicated time. The endogenous HER3 was immunoprecipitated and the amount of ubiquitination and total HER3 were detected using the anti-ubiquitin and HER3 antibodies, respectively. (C) Perhexiline-induced downregulation of HER3 is blocked by proteasome and lysosome inhibitors. MDA-MB-468 cells were treated with DMSO, MG132 (10 μM), or BFA1 (30 nM) in the absence or presence of perhexiline (10 μM) for 8 hours. The endogenous HER3 was detected by the anti-HER3 antibody. (D) Perhexiline induces the redistribution of endogenous HER3 to the lysosome. MDA-MB-468 cells treated with DMSO (i to iii) or 10 μM perhexiline (iv to vi) for 2 hours were fixed and analyzed using confocal microscopy. The localization of HER3 and the lysosome was visualized using the anti-HER3 antibody (green) and LysoTracker (red), respectively. Co-localization of HER3 (green) with lysosome (visualized by LysoTracker Red) was observed in perhexiline-treated cells as yellow puncta marked by arrows. DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; HER3, human epidermal growth factor receptor 3.