Piperlongumine

Piperlongumine (Selleck Chemicals, China) was dissolved in tween-80/water (10/90, v/v) at a final concentration of 0.5 mg/ml, and was administered to mice (5 mg/kg/d) daily via intraperitoneal injection according to corresponding therapeutic schedules. Glutathione (Selleck Chemicals, China) was dissolved in normal saline at a final concentration of 40 mg/ml, and was administered to mice (400 mg/kg/d) daily via intraperitoneal injection corresponding therapeutic schedules. Mannose (AbMole, China) was dissolved in 200 ml sterile water (20% (w/v)), which each mouse received a total amount of 40 g via normal drinking per week [13 (link)], supplemented with one dose of 40 mg by oral gavage three times per week. For αPD1 immunotherapy, αPD1 (clone RMP1-14, Bio X Cell) or its isotype control was administered to mice via intraperitoneal injection with a first dose of 400 µg/mouse and other doses of 200 µg/mouse every other day according to corresponding schedules (6 doses in total). Control animals received equivalent doses of isotype murine IgG according to the same dosing schedule.

Human CRC (HCT-116 and LoVo) and noncancerous gastric epithelial (GES-1) cells were provided by the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (China), and maintained in DMEM containing 10% heat-inactivated fetal bovine serum (FBS), and 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, USA). Oxaliplatin and piperlongumine were provided by Selleck Chemical (China). N-acetyl-l-cysteine (NAC) was manufactured by Beyotime Biotech (China). Antibodies targeting Bax, cleaved poly-ADP ribose polymerase (PARP), Bcl-2, phosphorylated histone 2AX (γH2AX), ATF4 (activating transcription factor-4), CHOP (CCAAT/enhancer-binding protein homologous protein), p-eIF2α, and eIF2α (eukaryotic initiating factor 2) were manufactured by Cell Signaling (USA). Anti-GAPDH primary and horseradish peroxidase-conjugated secondary antibodies were provided by Santa Cruz (USA). FITC-Annexin V apoptosis Detection Kit I and propidium iodide (PI) were manufactured by BD Pharmingen (USA).

‘The Spectrum Collection’ compound library (MicroSource Discovery Systems), was obtained at 10 mM in DMSO in 32 × 96-well plates with 80 compounds per plate. Upon arrival, plates were aliquoted and stored at −80 °C until use. Individual compounds were sourced from different suppliers (CAS/Product #). abcr GmbH: 1,4-Anthraquinone (635-12-1/AB177277). Santa Cruz Biotechnology, Inc: Scriptaid (287383-59-9/sc-202807). Selleck Chemicals LLC: Alantolactone (546-43-0/S8318); piperlongumine (20069-09-4/S7551); vorinostat (149647-78-9/S1047). Sigma-Aldrich/Merck KGaA: 2′,4′-Dihydroxy-4-methoxychalcone (81674-91-1/IDF00081); 4-hydroxychalcone (20426-12-4/S350664); chlorothalonil (1897-45-6/36791); DL-sulforaphane (4478-93-7/S4441); ethacrynic acid (58-54-8/E1800000); menadione (58-27-5/M9429); oxyphenbutazone (129-20-4/SML0540); papaverine hydrochloride (61-25-6/P3510); phenothiazine (92-84-2/P14831); thimerosal (54-64-8/T5125); thymoquinone (490-91-5/274666); ZPCK (26049-94-5/860794). Compounds were stored at −20 °C and stock solutions for tissue culture and C. elegans experiments were freshly prepared at 10–20 mM in DMSO.

The PI3K/AKT Inhibitor Piperlongumine (S7551) and AKT activator SC79 (S7863) were purchased from Selleck‐chem (Houston, TX, USA). Reagents were reconstituted and stored according to the manufacturer's instructions. Antibodies against MLH1 (ab124715), EGFR (#4108), p‐EGFR (ab56416), Her‐2 (BECN1, ab207612), PI3K (ab213521), AKT (#4668), Bcl‐2 (#4223), Bax (ab51520), active caspase‐3 (ab15580), and GAPDH (ab181602) were purchased from Cell Signaling Technology (Beverly, MA, USA) and Abcam (Cambridge, UK).