Supplementary Methods s6

Supplementary Methods

In vitro Enzyme assays

The inhibition of recombinant PI3Kβ, PI3Kα, PI3Kγ and PI3Kδ (AstraZeneca, UK) was evaluated in a

Kinase Glo based enzyme activity assay. Compounds in 100% DMSO were added to assay plates by acoustic dispensing. PI3K enzyme was added in a Tris buffer (50mM Tris pH7.4, 0.05% CHAPS,

2.1mM DTT, and 10mM MgCl2) and pre-incubated with compound for 20 minutes prior to addition of substrate solution containing PIP2 and ATP. The enzyme reaction was stopped after 80 minutes by the addition of Kinase Glo detection solution containing Luciferin and Luciferase. Plates were left for

30 minutes at room temperature then read on a Pherastar Instrument with a standard Luminescence filter block. The final concentrations of DMSO, ATP and PIP2 in the assay were 1%, 8μM, and 80μM respectively.

Growth factor stimulation and AZD8186 treatment of serum starved cells

PC3 and MDA-MB-468 cells were cultured to 70% confluence in complete medium in 6 well plates in

RPMI 1640 (Life Technologies, Inc) supplemented with 10% heat-inactivated FCS (Sigma) and 1% L-

Glutamine (Sigma). Cells were serum starved in RPMI 1640 overnight and following pre-incubation with vehicle (0.1% DMSO) or AZD8186 for 1 hour, stimulated for 15 minutes with 10 M LPA (1- oleoyl lysophosphatidic acid sodium salt stock solution, Tocris, R&D Systems) diluted in serum free medium (SFM) containing 1% delipidated BSA (Sigma). or 166ng/m EGF (Sigma) was prepared in the same medium. Western blot analyses for serum deprived cells treated with LPA or EGF , measured vinculin, phospho AKT-Ser473, total AKT, phospho ERK1/2 (Thr202/Tyr204) and Total ERK1/2 protein induced by the LPA /EGF stimulus in vehicle and AZD8186 treated serum starved cells. Briefly cell lysates were prepared in RIPA cell lysis buffer (Pierce) containing Mini Complete Protease Inhibitor

(Roche) and Halt Phosphatase Inhibitor (Pierce). BCA protein determination (Pierce) was carried out to load 20ug total protein in a volume of 25ul to each well of 4-12% Bis-Tris gels (Invitrogen Novex).

Assay procedures followed according to manufacturer protocols using CST antibodies for AKT and ERK and Sigma Aldrich antibody for Vinculin. Pierce Dura western blotting detection agent was used and signal measured on a Chemigenius system.

1 Cell Panel Proliferation assays

Culture conditions, source and identity testing of cell lines included in the large cell panel are provided in (Supplementary Table S1 Davies et al MCT 2012). Briefly, cells were seeded in 96-well plates, at a density to allow for logarithmic growth during the 72 hour assay, and incubated overnight at 37 oC, 5%

CO2 . Cells were treated with AZD8186 (30 to 0.003 M) for 72 hours and cell proliferation measured by MTS. The CellTiter Aqueous Non-Radioactive Cell Proliferation Assay (Promega) reagent was used in accordance with the manufacturer’s protocol and Absorbance measured with Tecan Ultra instrument. Pre-dose measurements were made and the concentration needed to reduce the growth of treated cells to half that of untreated cells (GI50) values were determined.

Histological analysis

Formalin-fixed, paraffin-embedded 4µm sections were baked on to slides at 37ºC overnight; then deparaffinised. Antigen retrieval used a RHS microwave vacuum processor (Milestone). A 2min cycle at 110ºC was used for phospho-AKT Thr308 (CST, 2965) and phospho-AKT Ser473 (Dako, M3628) antibodies, in EDTA (pH9) retrieval buffer. A 5 min cycle at 110ºC was used for phospho-PRAS40

(CST, 2997) in Citrate (pH6) buffer. A 2min cycle at 110ºC was used for Foxo3a (CST, 2497) in EDTA

(pH8) retrieval buffer. Endogenous peroxidase was blocked using 3% H 2O2 (or 0.18% H2O2 for Foxo3a staining) for 10min, followed by incubation with serum free block (Dako, X0909) for 20min to block non-specific binding. Primary antibodies were applied to slides (CST, 2965 [at 0.5µg/ml]; Dako, M3628

[at 2.8µg/ml]; CST, 2997 [at 0.75µg/ml]; and CST, 2497 [at 0.2µg/ml]) for 1h. All antibodies used were raised in rabbit and expression was detected using Rabbit Envision + System-HRP-labelled Polymer

(Dako), slides were incubated for 30min, followed by incubation with 3,3’-diaminobenzadine (DAB) chromagen (Dako) for 10min. Slides were then counterstained with Carazzi’s haematoxylin, dehydrated and mounted with coverslips.

Method for Supplemental Figures

MesoScale Phospho (Ser473), total AKT analysis.

2 Mesoscale Discovery Phospho (Ser473)/ Total AKT Assay kit was used to measure % phospho AKT protein induced by the LPA or EGF stimulus in vehicle and AZD8186 treated serum starved cells.

Briefly cell lysates were prepared in RIPA cell lysis buffer (Pierce) containing Mini Complete Protease

Inhibitor (Roche) and Halt Phosphatase Inhibitor (Pierce). BCA protein determination (Pierce) was carried out to load 20ug total protein in a volume of 25ul to each well of the 96W assay plate. Assay procedure followed according to manufacturer’s protocol and read on a SECTOR Imager 6000.

Cell Fractionation

LNCaP cells were treated with vehicle (0.1%DMSO) or 500nM AZD8186 for 2 hours. Cells were then extracted using a sub-cellular protein fractionation kit (Thermo Scientific kit 78840) to generate cytosolic, soluble nuclear and chromatin bound fractions. 15ug of total protein from each fraction was loaded and Western blotted for FOXO3a.