L). (D)Asiaticoside A site Expression of cytochrome c in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). Cells incubated with FITCconjugated isotype-specific antibodies were used as negative controls. ns: not significant compared to control. *p,0.05; **p,0.01; ***p,0.001 compared 25033180 to control. doi:10.1371/journal.pone.0059639.gwith 10 FBS, viable cells were counted by trypan blue dye exclusion. For tumor transfer, 56104 cells were suspended in 100 ml of phosphate buffered saline (PBS) and injected subcutaneously into the flank regions of mice. Ten to fourteen days after inoculation, the tumors became macroscopically apparent.Antitumor Activity: MacroscopyMice were inoculated with B16F10 melanoma cells as described above and were randomly allocated to four groups of 5 animals. On day 14 (counted from the initial inoculation date), the BNCT group was intraperitoneally (i.p.) injected with BPA (250 mg/Kg body mass), followed by 
thermal neutron irradiation. The irradiated control group did not receive BPA, but only the same thermal neutron irradiation. The control group received only i.p. saline solution. Tumor sizes were measured daily using a caliperlike instrument. The size measurement was converted into tumor volume by the equation: tumor volume = length 6 width2/0.52 [15]. Necropsies were performed 15 or 22 days after tumor inoculation (1 or 7 days after irradiation, respectively), according to the method of Dagrosa and co-workers [16]. The mice wereeuthanized by cervical dislocation and then 871361-88-5 web necropsied. Primary tumors were then resected and processed for histological examination. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Ethical Committee for Animal Research 
at the Butantan Institute (Permit Number: 479/09).RNA ExtractionTumor tissue collected in RNA later (Ambion) was fragmented in a tissue pulverizer (Mikro-Dismembrator U, B. Braun Biotech International, Melsungen, Hesse, Germany). The total mouse RNA was extracted from approximately 100 mg of tissue after homogenization, using the Trizol kit (Invitrogen Corporation, Carlsbad, CA, USA), in accordance with the manufacturer’s recommendations.Apoptosis in Melanoma Cells after BNCTFigure 5. Expression of extrinsic apoptotic markers in B16F10 melanoma cells and normal melanocytes (mean 6 s.d.) determined by flow cytometry. (A) Expression of TNF-R1 in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (B)Expression of TNF-R1 in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (C) Expression of cleaved caspase 8 in B16F10 melanoma cells after BNCT treatment and 16574785 neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (D)Expression of cleaved caspase 8 in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). Cells incubated with FITC-conjugated isotype-specific antibodies were used as negative controls. ns: not significant compared to control. *p,0.05; **p,0.01; ***p,0.001 compared to control. doi:10.1371/journal.pone.0059639.gQuantitative Real-time Poly.L). (D)Expression of cytochrome c in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). Cells incubated with FITCconjugated isotype-specific antibodies were used as negative controls. ns: not significant compared to control. *p,0.05; **p,0.01; ***p,0.001 compared 25033180 to control. doi:10.1371/journal.pone.0059639.gwith 10 FBS, viable cells were counted by trypan blue dye exclusion. For tumor transfer, 56104 cells were suspended in 100 ml of phosphate buffered saline (PBS) and injected subcutaneously into the flank regions of mice. Ten to fourteen days after inoculation, the tumors became macroscopically apparent.Antitumor Activity: MacroscopyMice were inoculated with B16F10 melanoma cells as described above and were randomly allocated to four groups of 5 animals. On day 14 (counted from the initial inoculation date), the BNCT group was intraperitoneally (i.p.) injected with BPA (250 mg/Kg body mass), followed by thermal neutron irradiation. The irradiated control group did not receive BPA, but only the same thermal neutron irradiation. The control group received only i.p. saline solution. Tumor sizes were measured daily using a caliperlike instrument. The size measurement was converted into tumor volume by the equation: tumor volume = length 6 width2/0.52 [15]. Necropsies were performed 15 or 22 days after tumor inoculation (1 or 7 days after irradiation, respectively), according to the method of Dagrosa and co-workers [16]. The mice wereeuthanized by cervical dislocation and then necropsied. Primary tumors were then resected and processed for histological examination. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Ethical Committee for Animal Research at the Butantan Institute (Permit Number: 479/09).RNA ExtractionTumor tissue collected in RNA later (Ambion) was fragmented in a tissue pulverizer (Mikro-Dismembrator U, B. Braun Biotech International, Melsungen, Hesse, Germany). The total mouse RNA was extracted from approximately 100 mg of tissue after homogenization, using the Trizol kit (Invitrogen Corporation, Carlsbad, CA, USA), in accordance with the manufacturer’s recommendations.Apoptosis in Melanoma Cells after BNCTFigure 5. Expression of extrinsic apoptotic markers in B16F10 melanoma cells and normal melanocytes (mean 6 s.d.) determined by flow cytometry. (A) Expression of TNF-R1 in B16F10 melanoma cells after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (B)Expression of TNF-R1 in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (C) Expression of cleaved caspase 8 in B16F10 melanoma cells after BNCT treatment and 16574785 neutron irradiation alone (irradiated control) compared to cells without any treatment (control). (D)Expression of cleaved caspase 8 in normal melanocytes after BNCT treatment and neutron irradiation alone (irradiated control) compared to cells without any treatment (control). Cells incubated with FITC-conjugated isotype-specific antibodies were used as negative controls. ns: not significant compared to control. *p,0.05; **p,0.01; ***p,0.001 compared to control. doi:10.1371/journal.pone.0059639.gQuantitative Real-time Poly.
