Ly294002

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).

For clonogenic assays, six-well tissue culture plates were seeded with 500 cells in complete medium. The medium was changed 24 h after cell seeding and replaced with complete medium containing the indicated concentrations of Binimetinib (Selleck), MK-2206 2HCl (Selleck), LY294002 (Calbiochem) or PLX4720 (Axon Medchem). After 9 days of incubation, colonies were fixed with 4% PFA (paraformaldehyde), stained with Crystal violet in 10% ethanol, and counted on images. IC50 were determined for each pharmacological agent and for each cell line using the number of colonies in mock treated condition as the top response. Cell lines have been treated with Binimetinib from 10⁻³ to 100 μM, with PLX4720 from 10⁻³ to 100 μM, with LY294002 from 5.10⁻³ to 50 μM and with MK-2206 from 10⁻³ to 10 μM. We used the resulted sigmoidal curves to calculate the IC50 with Graphpad Prism. Experiments were performed at least in triplicate.