The resulting plasmids were designated as WT1 − 17AA/− KTS −, + 17AA/− KTS −, − 17AA/+ KTS −, or + 17AA/+ KTS-pHR-SIN-CSGW dlNotI. We deleted the eGFP sequence in pHR-SIN-CSGW dlNotI and used this modified vector as a control vector. Preparation of infectious particles was performed according to established protocols [29]. PD0325901 in vivo All of the procedures involving animals in this study were approved by the animal care committee of Yamagata University in accordance with institutional and Japanese government guidelines for animal experiments. WT1 splice variants (− 17AA/− KTS, + 17AA/− KTS, − 17AA/+ KTS, and + 17AA/+ KTS) or control lentivirus vectors were co-transfected with packaging plasmids (pVSV-G

and pGag/pol) into 293 T cells to generate lentiviral particles. These particles were then used to transduce SKOV3ip1 cells, and cells stably expressing the vectors were selected with 1 μg/mL puromycin. SKOV3ip1 cells (2 × 106) expressing WT1 variants (− 17AA/− KTS [n = 13], + 17AA/− KTS [n = 13], − 17AA/+ KTS [n = 8], or + 17AA/+ KTS [n = 10]) or control vector (n = 8) were suspended in 250 μL PBS and inoculated by intraperitoneal injection (i.p.) into selleck inhibitor 5- to 7-week-old female BALB/CA nu/nu mice. Abdominal circumference and body weight were measured twice weekly. Mice injected with WT1 − 17AA/− KTS-expressing cells were euthanized with CO2 after 36 days and mice injected with cells expressing

control vector or the other variants were euthanized after 40 days to assess tumorigenecity by measuring volume of ascites, extent of dissemination, and weight of tumors. We used data from mice that were Wilson disease protein euthanized precisely after 36 or 40 days. In a second experiment, 2 × 106 SKOV3ip1 cells expressing the control vector or each WT1 variant were injected i.p. into 5- to 7-week-old female BALB/CA nu/nu mice (n = 30, with 6 mice per group of cells), and survival was evaluated from the first

day of inoculation until death. In a third experiment, SKOV3ip1 cells (2 × 106) expressing WT1 − 17AA/− KTS were implanted by i.p. into 5- to 7-week-old nu/nu nude mice (n = 10). After two week after inoculation, one group of mice (n = 5) was treated i.p with PBS twice weekly for 3 weeks. A second group of mice (n = 5) was treated ip with bevacizumab (5 mg/kg) twice weekly for 3 weeks. Bevacizumab was kindly provided by Chugai Pharmaceutical Co., Ltd. (Tokyo, Japan). Bevacizumab were diluted in 200 μl of PBS. Abdominal circumference and body weight were measured once a week. At 3 weeks after initiating treatment, mice were euthanized with CO2 to assess antitumor efficacy of bevacizumab by measuring volume of ascites and weight of tumors. Briefly, tumors were homogenized in RLT buffer. Total RNA was isolated and purified with an RNeasy− Mini kit (Qiagen, Valencia, CA, USA). cDNA was generated form 0.4 μg RNA using a QuantiTect Reverse Transcription kit (Invitrogen). cDNA (1.