Figure 4
Effect of recycling inhibition on cell surface EGFR upon EGF stimulation in RBE and MMNK-1 cells. (A) Left: cell surface EGFR before and after 1 hr of EGF stimulation without monensin treatment. Black columns: RBE; white columns: MMNK-1. Values are presented as the Mean Fluorescence Intensity (MFI) of cell surface EGFR.*p < 0.05. Middle: cell surface EGFR after 1 hr of EGF stimulation without or with monensin in RBE cells (black columns) and MMNK-1 cells (white columns). Values are presented as the percentage of the MFI of cell surface EGFR compared to that of RBE or MMNK-1 cells before EGF stimulation. *p < 0.05. Right: representative FACS data. Dark blue: cell surface EGFR before EGF stimulation without monensin; purple: cell surface EGFR after 1 hr of EGF stimulation without monensin; green: cell surface EGFR before EGF stimulation with monensin; light blue: cell surface EGFR after 1 hr of EGF stimulation with monensin. (B) EGFR (Alexa Fluor 488) and EEA-1 (Alexa Fluor 555) distribution after 1 hr of EGF stimulation without or with monensin in RBE and MMNK-1 cells as obtained by immunofluorescence. White arrows show points of colocalization. Scale bars: 10 μm. (C) Upper: suppression of Rab11a expression by siRNA. Lower: EGFR (Alexa Fluor 488) and EEA-1 (Alexa Fluor 555) distribution or EGFR (Alexa Fluor 555) and LAMP-1 (Alexa Fluor 488) distribution after 1 hr of EGF stimulation in mock- or Rab11a siRNA-transfected RBE cells as obtained by immunofluorescence. White arrows show points of colocalization. Scale bars: 10 μm. All results are representative of three or more independent experiments.