Fig. 1
Binding and induction of RON internalization by Zt/g4-DM1. a Levels of RON expression by BC and NSCLC cell lines. Individual BC and NSCLC cell lines (1 × 106 cells/ml) in 1 ml PBS in duplicates were incubated at 4 °C with 5 μg/ml of Zt/g4 for 60 min. Isotope matched mouse IgG was used as the control. Cell surface RON was quantitatively determined by immuno-fluorescence analysis using QIFKIT® (DAKO). The number of RON receptors was in a single cell was calculated according to the DAKO’s instruction. b Binding of DM1-conjugated Zt/g4 to cell surface RON. Individual BC or NSCLC cell lines at (1 × 106 cells/ml) were incubated at 4 °C with 5 μg/ml of Zt/g4-DM1 for 60 min followed by flow cytometric analysis. Control mouse IgG (CTL) and free Zt/g4 were used as the control. c The time-dependent RON internalization. BC and NSCLC cells (1 × 106 cells per dish) were treated at 37 °C with 5 μg/ml of Zt/g4-DM1, collected at different time points, washed with acidic buffer to remove Zt/g4 bound on the cell surface (31), and then incubated with 2 μg/mL of anti-RON mAb 2F2 [23]. Immunofluorescence was analyzed by flow cytometer using FITC-coupled anti-mouse IgG. The FITC-binding intensity from cells treated with Zt/g4-DM1 at 4 °C was set as 100 %. The IE50 values were calculated as the time required to achieving 50 % reduction of cell surface RON. d and e Immunofluorescent analysis of cytoplasmic RON: BC and NSCLC cells (1 × 105 cells per chamber) were treated at 4 °C or 37 °C with 5 μg/ml of Zt/g4-DM1 for 12 h followed by FITC-coupled anti-mouse IgG. CmIgG-DM1 was used as the control. After cell fixation, immunofluorescence was detected using the BK70 Olympus microscope equipped with a fluorescence apparatus. LAMP1 was used as a marker for protein cytoplasmic localization. DAPI was used to stain nuclear DNA