Supporting Materials and Methods

Supporting materials and methods

Drugs and compounds

The chemical structure of DC120 was showed in Fig.1A. DC120 was synthesized and initially dissolved in 100% DMSO to generate a 50mM stock solution and stored at

−20°C. U0126 and RAD001 were obtained from Selleck Chemical (Boston, U.S.A.), dissolved in 100% DMSO at high concentrations, and stored at -20°C.

Cyclophosphamide (CTX) was obtained from Jiangsu Hengrui (Lianyungang, China) and freshly prepared in normal saline (NS) prior to use. W7 was purchased from

Sigma-Aldrich (Samoa, U.S.A.) and dissolved in ddH2O to a 50mM stock solution. 3-

MA was purchased from Sigma-Aldrich and dissolved in PBS at 200mM stock solution. TG was purchased from Sigma-Aldrich and dissolved in 100%DMSO at

8mM stock solution. BAPTA-AM was purchased from Merck Millipore (Billerica,

U.S.A.) and dissolved in 100%DMSO at 10mM stock solution.

Antibodies and reagents

Primary antibodies of AKT1, phospho-AKT1 (Ser473), phospho-AKT1 (Thr308),

GSK3α/β, Caspase-3, poly (ADP-ribose) polymerase (PARP), ERK1/2 and phospho-

ERK1/2 (Thr202/Tyr204) and horseradish peroxidase–conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (California, U.S.A.).

Antibodies against FOXO3α, 4E-BP1, mTOR, P70S6K, PRAS40, phospho-FOXO3α

(Ser318/321), phospho-4E-BP1 (Thr37/46), phospho- mTOR (Ser2448), phospho-

P70S6K (Thr389), phospho-PRAS40 (Thr246) and phospho-GSK3β (Ser9), the AKT kinase assay kit and chemiluminescence reagents were obtained from Cell Signaling

Technology (Boston, U.S.A.). The Annexin V–FITC apoptosis detection kit was purchased from Calbiochem. The transferase-mediated FITC-12-dUTP nick-end labeling (TUNEL) Assay Kit was purchased from Roche. DMSO, DAPI and MTT were purchased from Sigma. Lipofectamine 2000 and Alexa Fluor–labeled anti- fluorescein antibodies were obtained from Invitrogen (NY, U.S.A.). Fluo-3/AM and

EGTA (0.5M solution) were purchased from Beyotime Institute of Biotechnology

(Jiangsu, China).

TUNEL assay

Frozen tumor sections were stained with 50µl TUNEL reaction mixture as recommended by the manufacturer (In Situ Cell Death Detection Kit, POD, Roche).

For the negative control, only 50µl labeling solution was added to each slide. The slides were incubated for 60min at 37°C in a humidified atmosphere in the dark, then counterstained with DAPI (1μg/ml) for 5min and mounted under glass coverslips.

Slides were observed using confocal microscopy with an excitation wavelength in the range of 450–500nm and detection in the range of 515–565nm (green). TUNEL- positive nuclei stained green, and all other nuclei stained blue.

Plasmid and siRNA

Adenoviral vector pMSCV-puro was purchased from Clontech Laboratories, Inc.

The HA-tagged AKT1 plasmid was constructed using standard cloning methods.

Restriction endonucleases were BglⅡ and Xhol. Upstream primer was AGA AGATC TACCATGGACTACAAGGACGACGATGACAAGATGAGCGACGTGGCTATTGT and downstream primer was ATACTCGAGTCAAGCGTAATCTGGAACATCGT

ATGGGTAGGCCGTGCCGCTGGCCGA.

The target sequence for AKT2-specific siRNA was CGUGGUGAAUACAUCAAG A

TT, The target sequence for AKT3-specific siRNA was GAGGAGAGAAUGAA UU

GUATT, CSNK1D-specific siRNA was GCACCUUGGAAUUGAACAATT,

DYRK1B-specific siRNA was CGGAGAUGAAGUACUAUAUTT, and ADCK3

-specific siRNA was CGGGACAAGUUGGAAUACUTT.