Herceptin Conjugate Induces Tumor Cell Necrosis Figure 6. Analysis of cell death mechanism. (a) Active caspase analysis. OS187 and SKBR3 cells were treated as described in Figure 2. Caspase-3, -8, and -9 activity were tested by CaspGLOW fluorescein active caspase staining kits. Red dot line, sham; black dash line, wild-type Herceptin (80 mg/ mL); black solid line, EDC-Herceptin conjugate (80 mg/mL). (b). Effect of caspase inhibitors on EDC conjugate-induced cell death. Z-DEVD-FMK, caspase 3 inhibitor. Z-IETD-FMK, caspase 8 inhibitor. Z-LEHD-FMK caspase 9 inhibitor. Z-VAD-FMK, caspase inhibitor. OS187 cells were treated with sham, EDC Herceptin conjugate 20 mg/mL or EDC Herceptin conjugate 20 mg/mL plus different caspase inhibitors at 30 mmol/L. (c) Effect of necrosis inhibitors on EDC-Herceptin conjugate-induced cell death. Necrostatin-1 (Nec-1), programmed cell necrosis inhibitor. OS187/SKBR3 cells were treated with sham, EDC-Herceptin conjugate 20/80 mg/mL, or EDC-Herceptin conjugate 20/80 mg/mL plus Nec-1 at 30 mmol/L. N = 5. *, P,0.05; **, P,0.01. doi:10.1371/journal.pone.0023270.g006 PLoS ONE | www.plosone.org 7 August 2011 | Volume 6 | Issue 8 | e23270