Herceptin Conjugate Induces Tumor Cell Necrosis Figure 5. Induction of late-stage apoptosis and necrosis by EDC-Herceptin conjugate treatment. (a) OS187 cells were treated with sham, wt Herceptin (wild-type Herceptin), or EDC conjugate (EDC-Herceptin conjugate) for 48 or 72 h and then stained with annexin v and PI to determine the necrosis and apoptosis under flow cytometry. Necrosis control, freeze cells in 280uC for 5 min, and then thaw cells at 37uC. Serum starving, serum starving overnight as apoptosis control. (b) SKBR3 cells were treated as described in Figure 3, followed by PI, annexin v staining, and flow cytometry analysis. doi:10.1371/journal.pone.0023270.g005 analyzed in both OS187 and SKBR3 cells after treatment. However, no difference was detected among sham, wild-type Herceptin, and EDC conjugate cells in both cell lines (Figure 6a). This flow cytometry result indicated that induction of cell apoptosis was not the main mechanism of EDC-Herceptin conjugate-mediated cell death. We confirmed this result by treating OS187 cells with EDC Hercpetin conjugate plus caspase 3 inhibitor, casepase 8 inhibitor, caspase 9 inhibitor or caspases PLoS ONE | www.plosone.org 6 August 2011 | Volume 6 | Issue 8 | e23270