Fig. 4

Endothelin A2 (Endo II) promotes human epidermal growth factor receptor 2 (HER2) internalization in response to epidermal growth factor (EGF) treatment of HER2-positive cancer cells. a Flow cytometry analysis of surface HER2 levels in HCC1954 pLKO vector (Vec) and Endo II knock-down (KD)1 cells treated with or without EGF (100 ng/ml) for 15 minutes (min) or 1 hour (h) and stained with HER2 affibody. A representative histogram (left) and mean fluorescence intensity (MFI) results from three experiments are shown (*P < 0.05, **P < 0.01, ***P < 0.001). b Representative confocal microscopy images are shown for HCC1954 pLKO Vec and Endo II KD1 cells treated with or without Texas Red-EGF (100 ng/ml) and HER2 affibody (green; DAPI counterstain also shown). Inserts were selected to highlight membrane (i) and internalized vesicle (ii) compartments. c Quantification of vesicles containing co-localized HER2 and EGF in HCC1954 pLKO Vec and Endo II KD1 cells treated for 15 minutes from 25 cells (results are representative of two independent experiments)