Collagen coated 24 well plates

LX-2 cells (Merck Millipore; Billerica, MA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific; Waltham, MA) supplemented with 2% fetal bovine serum (FBS) and 1% Pen/Strep (Omega Scientific; Tarzana, CA). Approximately 1 x 10⁶ cells were thawed in T-75 flasks (Corning Life Sciences; Corning, NY) containing 12 mL cell culture medium and placed at 37°C in a Hera Cell 5% CO₂ incubator (Thermo Fisher Scientific). Culture medium was replaced the first day after thawing, and then every 72 hours until 80% confluent. HepG2 and HEK293 cells (ATCC; Manassas, VA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) and 1% Pen/Strep (Omega Scientific). Culture medium was replaced the first day after thawing, and then every 48 hours until 80% confluent. Primary human hepatocytes (Thermo Fisher Scientific) were thawed in 50 mL Cryopreserved Hepatocyte Recovery Medium (CHRM) and plated in 500 υL William's E Medium supplemented with Hepatocyte Plating Supplement Pack on collagen-coated 24-well plates (Thermo Fisher Scientific). Culture medium was replaced the first day after thawing with William's E Medium supplemented with Hepatocyte Maintenance Supplement Pack.

PC12, a rat pheochromocytoma cell line, differentiates in the presence of forskolin and is used as a model of neurodegeneration. PC12 cells were seeded in collagen-coated 24-well plates (Thermo Fisher Scientific Inc.) at a density of 5.0 × 10³ cells/well (non-treated with chemotherapeutic agents) or 2.0 × 10⁴ cells/well (treated with chemotherapeutic agents). Neurite outgrowth was induced by 10 µM forskolin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) 24 h before exposure to drugs. The cells were treated with oxaliplatin (3 µM), cisplatin (10 µM), paclitaxel (3 nM), or bortezomib (300 nM) in the presence/absence of alogliptin (0.1–100 nM) or exendin-3 (9–39) amide (1 µM), the GLP-1 receptor antagonist, for 24 h. The concentrations of these drugs were determined based on previous reports^{30 (link)}. The living cells, which were not stained with trypan blue (Thermo Fisher Scientific Inc.), were analyzed using a phase-contrast microscope (CKX41; Olympus Co., Tokyo, Japan) after incubation. The neurite lengths were measured by ImageJ 1.51 software (Wayne Rasband, National Institutes of Health, MD, USA). Each experiment was performed in four wells per group.

APAP, nuclease free water and ethanol (Molecular grade) were purchased from Sigma-Aldrich (St. Louis, MO). miRNA and RNA isolation, cDNA synthesis and pre-amplification reagents were purchased from Qiagen (Valencia, CA). Collagen coated 24 well plates and phosphate buffered saline were obtained from ThermoFisher Scientific (Carlsbad, CA). The miR-122 mimic, miR-122 inhibitor, miR-378a mimic, miR-378a inhibitor, AllStars Negative control siRNA and Hs Cell Death Control siRNA were purchased from Qiagen (Valencia, CA).