Gdc 0068

T47D, MCF7 cells were purchased from American Type Culture Collection (ATCC) in 2012 - 2015. They were authenticated using STR testing and tested negative for Mycoplasma contamination. EFM19, BT474, MDAMB453, HCC202, MDAMB361, HCC1419, MDAMB415, HCC1937, CAL51, BT20, HCC1954, and JIMT1 cells were purchased from Cancer Cell Line Encyclopedia (CCLE) at the Broad Institute in 2015-2016, and were authenticated using SpectroCHIPII-G384 by Sequenom's MassARRAY Analyzer Compact. All the cells were maintained in RPMI-1640 with 10% fetal bovine serum. BYL719, GDC0941, BKM120, AZD1208, GDC0032, PI-103, BX795, BX912, MK2204, GDC0068, sirolimus, everolimus, PP242 and WYE were purchased from Selleck Chemicals (Supplementary Material and Methods). Blasticidine was purchased from Life Technologies. LGH447 was obtained from Novartis.

SUDHL4 and Jurkat cells were cultured in RPMI 1640 with L-glutamine and 25mM HEPES (Mediatech) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals), and 1% PenStrep, at 37 °C with 5% CO₂ in a humidified incubator, except TMD8 and HBL-1, which were cultured with 15% FBS. The SCARB1 blocking and rabbit IgG isotype control antibodies were obtained from Novus Biologicals. The PI3K inhibitor Pilaralisib (XL-147; Selleck Chem), the AKT inhibitor GDC-0068 (Selleck Chem), the SYK inhibitor R406 (Selleck Chem) and the BTK inhibitor ibrutinib (PCI-32756; Selleck Chem) were added to cells at the same time as HDL NP treatment. The cholesterol sequesterant, methyl-β-cyclodextran (MβCD; Sigma Aldrich), was added to cells at the same time as HDL NPs, with concentrations ranging from 156.25 µM to 10 mM.

Cells were plated in DMEM with 2% FBS in 96 well plates at 3,000 cells per well. 24 hours post-plating, cells were treated with a 9-point dose dilution of the indicated inhibitor, either as a single agent or in combination, with DMSO as a control. After 72 hours, viability was measured using [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) CellTiter 96 cell viability kit (Promega). Combination Indices (CI) were calculated using the Chou and Talalay method (24 (link)) with CalcuSyn software (CalcuSyn). Details of the cytotoxicity assays can be found in the Supplemental Methods. The therapeutic compounds used were as follows: OP449 (25 (link)) and DT1154 (16 (link)) were described previously. BEZ235, INK128, GDC-0068, PP242, CUDC101, VX680, XL880, and Dasatinib were purchased from Selleck Chem.

2.8 × 10³ MCF7, 5 × 10³ T47D or 5 × 10³ ZR-75–1 cells were plated in 96-well plates in CTR medium. After 24 h, medium was removed and cells were washed with PBS and incubated in either CTR (10% FCS and 1g/L glucose) or STS (1% FBS, 0.5 g/L glucose) medium. After a further 24 h, cells were stimulated with or without with tamoxifen or fulvestrant at the indicated concentrations. Where indicated, GDC0068, AZD5363 and LY294002 (from SelleckChem) were used at 1 μM, 400 nM and 2 μM concentration, respectively. Viability was determined 72 h later by CellTiter 96 Aqueous One assay (Promega) according to the manufacturer’s instructions.

Calyculin A, staurosporine and LY294002 were purchased from Calbiochem. OSU-03012 was acquired from Cayman Chemical Co. Torin 1 was purchased from Tocris, and GDC-0068 was acquired from Selleckchem. Insulin and cyclohexamide were purchased from Sigma. PZ09 was a kind gift from Dr Sourav Ghosh and Dr Christopher Hulme. The following antibodies were purchased from Santa Cruz Biotechnology: anti-p410 PKCζ (sc-12894-R), anti-total PKCζ (sc-216), anti-rpL23a (sc-130252) and anti-GST (sc-138). Antibodies for p308 Akt (9275), p450 Akt (9267), p473 Akt (4060), total Akt (9272), PDK1 (3062), GAPDH (2118), mTOR (2983), rpS6 (2317) p389 ribosomal protein S6 kinase (S6K) (9205), total S6K (9202) and GFP (2555) were purchased from Cell Signaling Technology. The anti-p560 PKCζ antibody was purchased from AbCam (ab62372), the anti-haemagglutinin (HA) antibody was from Covance (MMS-101P) and the anti-actin antibody was from Sigma (A2228). HRP-conjugated goat anti-mouse IgG and goat anti-rabbit IgG were from Calbiochem (401215 and 401315). AlexaFluor 488-conjugated goat anti-rabbit IgG was from Invitrogen (A110034). Phosphatidylserine (PS), PIP₃ and phosphatidylinositol-4,5-biphosphate (PIP₂) were purchased from Avanti Polar Lipids (840034, 850457, 850156 and 850155, respectively). Phosphatidic acid (PA) was purchased from Santa Cruz Biotechnology (201059).