Xl 765

Female BALB/c nude mice, aged 5-week, were raised in a sterile environment, and then, the 3 × 10⁶ A2780 cells transfected with stable H19-sh and its control plasmid were suspended in 100 μL phosphate buffer and subcutaneously injected into the dorsal subcutaneous of nude mice. For PI3K inhibition, mice were orally administrated with 50 mg/kg XL-765 (Selleck) once per day [19 (link)]. The tumor growth of 5 nude mice in each group was detected every 7 days and worked out by the formula of volume = length^∗ width^2∗0.5². Twenty-eight days after injection, the mice were euthanized and the size and mass of the tumor were accurately measured. The animal study was reviewed and approved by Affiliated Hangzhou First People's Hospital, Zhejiang University School of Medicine.

Embryos were placed 5 per well in 48-well plates containing 400 µl of embryo medium/0.1% DMSO. Test compounds were added directly to the embryo medium. For intersegmental vessel (ISV) angiogenesis assay, embryos were treated from 6–24 hpf and from 2–5 dpf for hyaloid vessel (HV) angiogenesis assay [17] (link). Larvae were left to incubate with drugs at 28°C on a 14 h light/10 h dark cycle, euthanized and fixed with 4% paraformaldehyde (PFA) in PBS at room temperature for 2 hours before analysis. Drugs were purchased from the following: Rapamycin (Selleckchem S1039), NVP-BEZ235 (Selleckchem S1009), PI-103 (Selleckchem S1038), LY294002 (Sigma L9908), A66 (Selleckchem S2636), WYE125132 (Selleckchem S2661), Palomid 529 (Selleckchem S2238), XL147 (Selleckchem S1118) and XL765 (Selleckchem S1523). 2–5 dpf zebrafish larvae were euthanized in ice-cold PBS, or 4% PFA, or lysis buffer.