Pf 04691502

The activity of the PI3K/Akt signalling pathway was evaluated by specific phospho-ELISA kits and confirmed by 2-DE analysis combined with WB using an anti-Akt phospho-substrate (RXXS*/T*, Cell Signalling Technology, Danvers, MA, USA), as previously reported [71 (link),72 (link)]. Synchronized cells were treated for 2 h with the PI3K inhibitor LY294002 (10 µM, Selleck Chemicals, Houston, TX, USA) or the dual PI3K/mTOR inhibitor PF04691502 (1 µM, Merck) and apoptosis was evaluated in C91/PL, C91/III, BJAB and Jurkat cells 24 and 48 h after treatment by flow cytometry after Annexin V/PI staining (Annexin-V-FLUOS Staining Kit, Merck). Two-fold dilutions of the two inhibitors were used to analyse the sensitivity of the four cell lines by MTT assay. Specifically, synchronized cells were seeded in 96-well plates (3 × 10⁴/well) and treated with LY294002 (from 2 μM to 0.25 μM) and PF04691502 (from 20 μM to 2.5 μM) for 48 h. IC₅₀ was determined by a nonlinear regression analysis using the GraphPad Prism software (6.07 for Windows, San Diego, CA, USA).

Briefly, A2780-SP cells (2 × 10³) were seeded in 6-well ultra-low attachment plates (Corning) with or without 10 μM PF-04691502 (Sigma). After 7 days, the number of tumor spheroids was determined, and images were acquired by using EVOS 7500 machine (Thermo Scientific). The experiments were repeated three times.