DNA fragmentation Analysis and sub-G1 DNA content TUNEL analysis was performed to determine DNA fragmen- tation in FEMX and Melmet-5 cells as previously described [11]. The Melmet-5 cells were pre-treated with Z-VAD-FMK (50 mM)+Z-FA-FMK (50 mM) for 45 min. FEMX and Melmet-5 cells were subsequently treated with 9.2.27PE6ABT-737 for 24 h. The experiments were repeated at least three times. Quantitation of dead cells by measurement of sub-G1 DNA content using propidium iodide assay was carried out. FEMX, Melmet-5 and MelRM cells were treated with ABT-737 for 24, 32 and 48 h, harvested, washed with PBS, fixed in icecold MeOH and kept at 220uC until analysis. The cells were washed twice in PBS before subjected to PI (5 mg/ml) and RNase and analyzed on a BDFACSCantoTM (Becton Dickinson, San Jose, CA). The experiment was repeated at least twice for each time point. Intracellular calcium levels FEMX, Melmet-5, MelRM, MelRMshCtr and MelRMshMcl-1 cells were treated with 9.2.27PE6ABT-737 for 12, 16 or 24 h. Cells were collected, washed with PBS, incubated with 1 mM Calcium GreenTM-1 (Molecular Probes) for 30 min, washed with PBS and subjected to flow cytometric analysis on a Becton Dickinson FACSCantoTM (Becton Dickinson). Intact cells were gated in a forward and side scatter analysis, and results are reported in median fluorescence intensity of the intact cells. Untreated cells were set to the given value 1, and values for the treated cells were calculated accordingly. The experiment was repeated at least three times for each time point per cell line. Analysis was carried out on ,10.000 cells per sample. Ethics Statements All procedures and experiments involving animals were approved by The National Animal Research Authority and carried out according to the European Convention for the Protection of Vertebrates used for Scientific Purposes. Nude female mice (BALB/c (nu/nu)) were bred in our nude rodent facility. The animals were kept in a specific pathogen-free environment, in positive pressure rooms with filtered and humidified air. The animals were kept under standard conditions, and food and water were supplied ad libitum. In vivo studies Nude female mice with Melmet-5 xenografts in the right and left flanks were used. Tumors were size-matched with an average volume of 150–161 mm3/group. The animals were divided into four groups; control, 9.2.27PE, ABT-737 and 9.2.27PE+ABT-737. Treatment was initiated with 10–12 tumors per group and mean body weight #31 g. ABT-737 (100 mg/kg) was administered once daily on day 1–5 and 15–19 by intraperitoneal injection. The 9.2.27PE immunotoxin (31.25 mg/kg) was given intravenously 5– 6 h after ABT-737 treatment on day 1 and 15. All animals administered ABT-737 developed skin rash, no other adverse effects were observed. Two animals in the 9.2.27PE6ABT-737 group developed skin ulcers and were sacrificed on day 8 and 21. For the second experiment, the concentration of ABT-737 was decreased to 50 mg/kg and administered on day 1–5 and 15–19. The 9.2.27PE immunotoxin (31.25 mg/kg) was given 5–6 h after ABT-737 treatment on day 1 and 15. Treatment commenced when the mean tumor volume reached 92–102 mm3/group and the mean body weight #31 g. For unknown reasons, two mice died shortly after treatment start in the combination group, and were not included in the final analysis. One mice died for unknown reasons on day 15, no other adverse events were 9.2.27PE and ABT-737 in Melanoma recorded. Tumor size was measured 1–3 times weakly by caliper measurements and tumor volume was calculated (tumor volu- me = L6W2/2). Tumor volume was plotted against time (days). Body weight was recorded throughout the experiments. ANOVA was used to calculate statistical differences between groups. Results with a p value less than 0.05 were regarded as significant. Results 9.2.27PE and ABT-737 reduced cell viability in a panel of melanoma cells The 9.2.27PE induced a time- and dose dependent decrease in cell viability in the FEMX, Melmet-1, Melmet-5, Melmet-44, MelRM and MM200 cells (Fig. 1A). The effect of 9.2.27PE was consistent in the five cell lines, as opposed to the effect of ABT- 737. ABT-737 IC50 was obtained in MelRM and MM200 after 24 h (Fig. 1B and C). With increased exposure time to 48 h, IC50 was also obtained in FEMX and Melmet-1 cells. Melmet-5 and Melmet-44 cell proved to be less sensitive to ABT-737 with IC50.20 mM after 48 h treatment. The sensitivity of the cells to 9.2.27PE or ABT-737 was not related to the BRAF status of these cell lines (Fig. 1C). Furthermore, 24 h treatment with the 425.3PE immunotoxin (100 ng/ml) targeting the epidermal growth factor receptor (EGFR), the antibody 9.2.27 (100 ng/ml) or the PE (100 ng/ml) did not result in decreased cell viability in MelRM cells (data not shown). These results are consistent with results previously obtained for the FEMX cells, emphasizing the specificity of the 9.2.27PE immunotoxin [11]. 9.2.27PE in combination with ABT-737 synergistically induce apoptosis To investigate whether the combination of 9.2.27PE and ABT-737 could induce synergistic cytotoxicity in melanoma cells, the cells lines were exposed to different concentrations of the two drugs for 24 and 48 h. The CalcuSyn software program was used to calculate synergistic, additive or antagonistic effects. Combining 10 ng/ml 9.2.27PE+1 mM ABT-737 or 100 ng/ml 9.2.27PE+10 mM ABT-737 caused combination index (CI) values in the 0.59 to 0.003 range, indicating synergy to very strong synergy, after 24–48 h (Fig. 1D and Table S1) [23]. As seen in Figure 2A, 9.2.27PE+ABT-737 caused clear cytotoxic effect in both FEMX and Melmet-5 cells, with morphologically rounding of the cells and detachment from the surface of the flasks, compared to single agent treatments with 9.2.27PE or ABT-737, where only a few cells had started to round up. Similar effects were seen in MelRM cells (data not shown). As both FEMX and MelRM cells proved to be sensitive to the ABT- 737 (Fig. 1B), they were further treated with A-793844, the enantiomer and negative control of ABT-737. With concentra- tions up to 10 mM, no decrease in cell viability was observed after 24 h or 48 h (48 h: FEMX cells only) (Figure S1). The cytotoxic effect observed when combining 9.2.27PE and ABT-737 was associated with apoptosis, as enhanced PARP inactivation and activation of caspase-3 was observed in both FEMX cells and Melmet-5 cells after 24 h treatment (Fig. 2B and C). Pre-treatment of the Melmet-5 cells with the pan-caspase inhibitor Z-VAD-FMK or the cathepsin B/L inhibitor Z-FA- FMK resulted in a shift upwards of the caspase-3 band from p17 to p19, indicating that the active caspase-3 was blocked (Fig. 2C) [24]. In addition to inhibiting the inactivation of PARP, the combination of the two inhibitors suppressed the processing of the procaspase-3 protein. Furthermore, pre-treatment of the FEMX cells with the combination of Z-VAD-FMK and Z-FA-FMK, inhibited the synergistic cytotoxic effect caused by 9.2.27PE PLoS ONE | www.plosone.org 3 September 2011 | Volume 6 | Issue 9 | e24012