Cleaved casepase 3

BEZ235 was purchased from Selleck, dissolved in dimethyl sulfoxide (DMSO) and stored at −20 °C. Chloroquine (CQ) was purchased from Sigma, dissolved in sterile double distilled water, and stored at −20 °C. 3-methyladenine (3-MA) was purchased from Selleck, dissolved in PBS and stored at −20 °C. Z-VAD-FMK was purchased from Beyotime and stored at −20 °C. The human CML cell line K562 was obtained from the Fujian Institute of Hematology, and the KBM7R cell line was purchased from the Harbin Institute of Hematology. Fetal bovine serum and RPMI 1640 medium were purchased from Gibco. Annexin V-FITC/PI kit was purchased from Becton Dickinson. MTS kit was purchased from Promega. Primary antibody AKT, P-AKT, S6K, P-S6K, Cleaved casepase-3, LC3I/II and HRP-labeled goat anti-rabbit IgG were purchased from Abcam. The RFP-GFP-LC3 double-labeled adenovirus was purchased from Hanbio. Cell constant temperature incubator (Thermo, USA), Refrigerated centrifuge (Eppendorf, Germany), Infinite M200 microplate reader (Tecan, Switzerland), FACS Calibur TM flow cytometry (Becton Dickinson, USA). Western blot electrophoresis, Semi-dry meter and Gel imager (Bio-Rad, USA). Hu-12A Transmission electron microscope (Hitachi, Japan), Laser scanning confocal microscope (Zeiss, Germany).

Total protein was extracted from tissue and cells using RIPA buffer with phenylmethanesulfonyl fluoride protease inhibitor. Protein concentration was determined with bicinchoninic acid (BCA) assay kits (Thermo Fisher). The proteins were separated by 10% or 12% Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk, incubated with primary antibodies overnight at 4°C and then with the designated secondary antibody at room temperature for 1 h after washing with once with tris-buffered saline and Polysorbate 20 (TBST). The signals were detected with an enhanced chemiluminescence (ECL)-Plus kit (Amersham Biosciences). Primary antibodies against the following proteins were used: KLF13 (1:1,000; Proteintech, 18352-1-AP), GAPDH (1:7,000; Proteintech, 10494-1-AP), HMGCS1 (1:1,000; Proteintech, 17643-1-AP), E-cadherin (1:5,000; Proteintech, 20874-1-AP), N-cadherin (1:6,000; Proteintech, 22018-1-AP), vimentin (1:5,000; Abcam, ab92547), CDK2 (1:2,000; Proteintech, 10122-1-AP), CDK4 (1:2,500; Proteintech, 11026-1-AP), cyclinD1 (1:1,500; Proteintech, 26939-1-AP), cleaved-casepase-3 (1:500; Abcam, ab32042), and KLF13-flag (1:50; Cell Signaling).