P ulk

Cell culture medium, DMEM, RPMI-1640, trypsin, phosphate-buffered saline (PBS), and FBS were purchased from Gibco (CA, United States). DMSO, 3-Methyladenine (3-MA), Compound C (HY-13418), and AMPKinone (HY-12831) were products of MedChem Express (NJ, United States). Antibodies against AMPK, p-AMPK, mTOR, p-mTOR, ULK, p-ULK, Beclin-1, and LC3I/II were obtained from Cell Signaling Technology (Danvers, MA, United States). Horseradish peroxidase-conjugated secondary antibody (IgG goat anti-rabbit or anti-mouse) and beta -tubulin antibody were purchased from CWBIO. BCA protein assay kit was product of Beyotime (Shanghai, China). MTS assay kit was purchased from Promega (Promega, WI, United States). All other chemicals used were the highest purity obtained from commercial sources.

HCT116 and SW620 cells were lysed by cell lysis solution (1× PBS, 1% Nonidet P40, and 1 mM EDTA) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). Equal amounts of protein were diluted in 2× sodium dodecyl sulfate (SDS) loading dye, separated on 8–15% SDS-polyacrylamide (SDS-PAGE) gels, and transferred onto nitro-cellulose membranes (GE Healthcare Life Sciences, Boston, MA, USA). After blocking with 5% skim milk at room temperature for 1 h, the membranes were probed overnight with primary antibodies at 4 °C. Anti-O-GlcNAc antibody was purchased from Sigma-Aldrich. An anti-PARP antibody was purchased from Invitrogen. Anti-cleaved caspase-3, IRE1α, P-eIF2α, eIF2α, CHOP, P-AMPK, AMPK, P-ULK, P-mTOR, mTOR, P-p70 S6K, p70 S6K, ATG5, and LC3B antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ULK, PERK, ATF6, ATF4, P-JNK, JNK, Bax, P-Bcl2, Bcl2, p62, GFAT, caspase-8, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were washed with TBST and incubated with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit immunoglobulin G secondary antibodies at room temperature for 30 min. Immunoreactive bands were developed using an ECL Plus Detection System (GE Healthcare Life Sciences) and the bands were quantified using ImageJ software (NIH, USA).

TRIzol, LPS and phenylmethanesulfonyl fluoride (PMSF) were purchased from Sigma (Saint Louis, MO, USA). Deh was purchased from Shanghai Yuanye Bio-Technology Co. CCK8 was purchased from Saint-Bio Co. (Shanghai, China). Compound C (CC) and 3-methyladenine (3-MA) were purchased from Selleckchem (Shanghai, China). p-Beclin, Beclin, p-ULK, ULK, AMPK, p-AMPK and LC3B were purchased from Cell Signaling Technology (Boston, MA, USA). P62 was purchased from Proteintech (Rosemont, IL, USA). β-Tubulin was purchased from Bosterbio in Dallas (Pleasanton, USA). IgG was purchased from Beyotime (Shanghai, China). HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from Bosterbio.