Oxaliplatin

The Annexin V-PE and 7-AAD (BD Biosciences, San Jose, CA, USA) double staining method was used to examine cell viability. The frequency of apoptosis was measured using the BD FACSCalibur™ Flow Cytometer (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Before assessing the chemosensitivity of colon cancer cells to oxaliplatin, the optimal drug concentration of oxaliplatin inducing cell death was determined on untransfected cells. Cells were treated with oxaliplatin (MedChem express, Monmouth Junction, NJ, USA) at a final concentration of 50 μg/mL for 24 h. The percentage of oxaliplatin-treated and untreated apoptotic cells was calculated according to the number of cells positive or negative for Annexin V-PE and 7-AAD. Results are presented as the percentage of total cells that were living cells (Ann−/7-AAD-), early apoptotic cells (Ann+/7-AAD-), late apoptotic cells, and dead cells (Ann+/7-AAD +).

CRC cell HCT116 was purchased from Cell bank of the Chinese Academy of Sciences (Shanghai, China). Oxaliplatin (MedChemExpress) was used to establish Oxaliplatin‐resistant cell line (HCT116‐OxR) as previously described.^[26, ^27] Cells were cultured in RPMI1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO₂ and 95% air.

SCFAs were obtained from Sigma-Aldrich (St. Louis, MO, USA). Erastin, ferrostatin-1, FIN56, necrostatin-1, oxaliplatin, RSL3,pertussis toxin, Entinostat, Ricolinostat, Nicotinamide, SIS17, T5224 and TMP269 were purchased from MedChemExpress (Shanghai, China). DCFH-DA, Hoechst33342, N-acetyl-l-cysteine, propidium iodide, trichostatin A, and Z-vad-FMK were obtained from Beyotime (Shanghai, China). Other cytokines/chemicals included B27 (Invitrogen, Carlsbad, CA, USA), recombinant human EGF and FGF (PeproTech, Rocky Hill, NJ, USA), Cystine (Solarbio, Beijing, China), C11-BODIPY 581/591 dye (Abclonal, Wuhan, China).

Oxaliplatin (MedChem Express, Monmouth Junction, NJ) was dissolved at 0.6 μg/μl in 0.9% NaCl. RgIA4 was dissolved at 0.01 μg/μl in 0.9% NaCl. Mice were injected i.p. daily with Oxaliplatin (3.5 mg/kg) or 0.9% saline (vehicle). Additionally, mice were injected s.c. with RgIA4 (40 μg/kg) or vehicle. Treatment weeks included injection on Thurs, Fri, and Mon–Wed. On Thurs, 24 h after last injection, mice were assessed for cold allodynia. Injections stopped on day 21, with assessment occurring on day 22 and subsequent testing occurring once a week for three additional weeks. Testing was conducted using a cold plate test chamber (IITC, Inc Life Science, Woodland, CA). Animals were allowed to acclimate in the chamber at room temperature (23°C) for 5 min. Temperature was then lowered at a rate of 10°C per minute. The testing was stopped when the animal lifted both forepaws and shaking or licking occurred. Alternating lifting of forepaws was not scored. Throughout the study period, experimenters were blinded as to the identity of the injected compounds.

Human SHSY-5Y neuronal cells were purchased from the Type Culture Collection of the Chinese Academy of Sciences (TCCCAS, Shanghai, China) and cultured in the DMEM completed medium supplemented with 10% FBS and CO₂ at 37°C. The cells were recovered in a 150 mm cell culture dish and split by trypsinization until 90% confluence was reached. For the experiment, 2 × 10⁵ cells /well were cultured in a 6-well plate and treated with 50 μM oxaliplatin (Cat#HY-17371, MedChemExpress, USA) [4 (link),5 (link)] in the presence or absence of Memantine (Cat#1189713-18-5, Chemgen bio-Tech Pioneer, USA) [17 (link)] at the concentrations of 5 or 10 µM for 24 hours. In this study, cells from passages 3 to 6 were used for experiments.

The following chemical reagents were used in this study: Oxaliplatin (#HY‐17371; MedChemExpress, NJ, USA), 5‐Aza‐dC (#A3656; Sigma–Aldrich, MO, USA), Trichostatin A (#V900931; Sigma–Aldrich), chloroquine (#HY‐17589A; MedChemExpress), cycloheximide (#S7418; Selleck Chemicals, TX, USA), MG132 (#M7449; Sigma–Aldrich), RO‐3306 (#S7747; Selleck Chemicals), erastin (#S7242; Selleck Chemicals), RSL3 (#S8155; Selleck Chemicals), ferrostatin‐1 (#S7243; Selleck Chemicals), deferoxamine (#HY‐B1625; MedChemExpress), N‐acetyl‐cysteine (#HY‐B0215; MedChemExpress), Z‐VAD‐FMK (#V116; Sigma–Aldrich), necrostatin‐1 (#S8037; Selleck Chemicals), Rosiglitazone (#HY‐17386; MedChemExpress), and liproxstatin‐1 (#HY‐12726; MedChemExpress).