Following drug treatment, media was aspirated and cells were fixed in 100 μl of 4% formaldehyde for 15 min at room temperature before being washed three times in PBS. Cells were permeabilized with 100 μl of ice-cold methanol for 10 min at −20 °C and again washed in PBS. Staining was performed by blocking for 60 min at room temperature (5% goat serum/0.3% Triton X-100 in PBS) PI3K inhibitors in clinical trials after which primary antibody incubations (in 1% BSA/0.3% Triton X-100 in PBS) were carried out overnight at 4 °C. Antibodies to pAKT (Cell Signalling; #9271 at 1:50) and total AKT (Cell Signalling; #2920 at 1:50, were

optimized for InCell western incubations. Secondary mTOR activity antibody detection was carried out as described for western blot analysis with 1:800 IRdye680 (for the normalizer) and 1:800 of IRdye800 (for the target). Analysis was carried out after pAKT: tAKT normalisation. Denatured and reduced protein lysates were

spotted onto nitrocellulose-coated glass slides (Whatman, Stamford, ME) using a MicroGrid II robotic spotter (DigiLab, Holliston, MA) as previously described (Spurrier et al., 2008). Three replicates were spotted per sample in five two-fold dilutions (resulting in a total of 15 spots per sample). Slides were hydrated in Li-Cor blocking buffer for 1 h (LI-COR Biosciences, Nebraska, USA), and then incubated with primary antibodies overnight at 4 °C in a sealed box containing a damp paper

towel. Antibodies to pAKT (Cell Signalling; #9271 at 1:50), and PP2A (Cell Signalling; #2259 at 1:50), were optimized for RPPA incubations. Slides were stained using matched total and phospho-proteins duplexed on each slide. The following day slides were washed three times in PBS/0.1% Tween no 20 (PBS-T) at room temperature for 5 min before incubating with far-red fluorescently-labelled secondary antibodies diluted in Li-Cor Blocking Buffer (1:2000) at room temperature for 45 min with gentle shaking. Slides were then washed in excess PBS/T (x3)/PBS (x3) and allowed to air dry before reading on a Li-Cor Odyssey scanner at 680 nm and 780 nm. RPPA analysis was performed using MicroVigene RPPA analysis module (VigeneTech, Carlisle, MA, USA). Spots were quantified by accurate single segmentation, with actual spot signal boundaries determined by the image analysis algorithm. Each spot was quantified by measuring the total pixel intensity of the area of each spot (volume of spot signal pixels), with background subtraction of 2 pixels around each individual spot. The quantification y0 (intensity of curve) or rsu (relative concentration value) of sample dilution curves were normalised using the corresponding total protein.