The levels of IGFBP-3 are responsible to Nimotuzumab-enhanced radiosensitivity of EGFR overexpressing ESCC cells. (a) KYSE30 and TE-1 cells were deprived of serum overnight and then incubated first for 3 hours with or without Nimotuzumab and then for an additional 15 minutes in the additional absence or presence of EGF (100 ng/mL). Cell lysates were evaluated for IGFBP-3, phosphorylated and total EGFR by Western blot. (b) IGFBP-3 silenced KYSE30 cells were harvested after 72 hours exposure to Nimotuzumab (100 μg/mL) or not. The expression levels of IGFBP-3 in control (non-silenced cells), IGFBP-3-silenced and Nimotuzumab-treated IGFBP-3-silenced cells were determined by Western blot. The experiments were performed in triplicate. (c) IGFBP-3-silenced KYSE30 cells were treated with RT (10 Gy) in presence or absence of Nimotuzumab (100 μg/mL) for 72 hours. All cells were collected for staining with annexin-V/PI, and cell death was measured by flow cytometry. In RT experiment, the radiosensitivity of KYSE30 cells was significantly inhibited after IGFBP-3 silence, and the cell death rates were reduced from 46.2 ± 2.1% to 25.9 ± 3.1% (P < 0.05). After the administration of Nimotuzumab, the RT response of IGFBP-3-silenced KYSE30 cells was not enhanced (the cell death rate was 28.6 ± 2.8%) (P > 0.05). The experiments were performed in triplicate.