Fig. 2.

Effect of H-Zt/g4-MMAE on RON internalization, cell viability, and death: (a) H-Zt/g4-induced cell surface RON internalization. PDAC cell lines BxPC-3, FG and L3.6pl (1 × 10⁶ cells per dish) were treated at 37 °C with 5 μg/ml of H-Zt/g4-MMAE, collected at different time points, washed with acidic buffer to eliminate cell surface bound IgG [31], and then incubated with 2 μg/mL of anti-RON mAb Zt/c1 [34]. Immunofluorescence was analyzed by flow cytometer using FITC-coupled anti-mouse IgG. Immunofluorescence from cells treated with H-Zt/g4 at 4 °C was set as 100%. Internalization efficiency (IC₅₀) was calculated as the time required achieving 50% reduction of cell surface RON. (b) Intracellular localization of internalized RON. FG cells in a 6-well plate were treated with 5 μg/ml of H-Zt/g4 at 4 °C or 37 °C for 12 h followed by mouse anti-human IgG1-coupled with FITC. Nuclear DNAs were stained with DAPI. LAMP-1 was used as a marker for protein cytoplasmic localization. Similar results also observed in additional three PDAC cell lines (data not shown). (c) Effect of H-Zt/g4-MMAE on viability of PDAC cells. Three PDAC cell lines (8000 cells per well in a 96-well plate in triplicate) were treated with different amounts of H-Zt/g4-MMAE for 96 h. Panc-1 cells served as the negative control. Cell viability was determined by the MTS assay. (d) Death of PDAC cells after H-Zt/g4-MMAE treatment. PDAC cells were treated with different amounts of H-Zt/g4-MMAE for 96 h. The percentages of cell death were determined by the trypan blue exclusion method. Data shown in (c) and (d) are derived from one of three experiments with similar results