F 12 nutrient mixture ham

Ginsenoside panaxatriol was obtained from Must Bio-Technology (Chengdu, China). PTX was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM) (11995-040), F-12 nutrient mixture (Ham) and fetal bovine serum (FBS) were bought from Life Technologies (Grand Island, NY, USA). MammoCul medium (human) and supplements were purchased from STEMCELL Technologies (Vancouver, BC, Canada). CellTiter-Glo luminescent cell viability assay kits were purchased from Promega Corporation (Madison, WI, USA). iScript gDNA Clear cDNA Synthesis Kits and iTaq Universal SYBR Green Supermix Kits were purchased from Bio-Rad Laboratories (Hercules, CA, USA). p-IRAK1 S376, IRAK1, p-P65 S536, P65, p-ERK1/2, ERK1/2, BAX, BCL-2 and MCL-1 antibodies were supplied by Cell Signaling Technology (Danvers, MA, USA). Beta-actin antibody was purchased from Sigma–Aldrich (St. Louis, MO, USA).

Fetal calf serum (FCS), Dulbecco’s modified Eagle’s medium (DMEM), and F-12 nutrient mixture (HAM) were obtained from Invitrogen Corp. (Carlsbad, CA). The anti-TLR2 IgG clone T2.5 neutralizing antibody (MAb-mTLR2) and isotype mouse IgG1 were provided by Invivogen (San Diego, CA). The anti-His tag antibody (Ab18184) was purchased from Abcam (Cambridge, MA). All other chemicals were supplied by Sigma (St. Louis, MO).

Agarose micro-well arrays were made using locally developed Teflon stamps and low melting point agarose (Sigma-Aldrich). The agarose, 40 g L⁻¹, was dissolved in phosphate buffered saline (PBS) at 100 °C and pipetted into the culture ware. The Teflon stamps were pressed into the agarose solution for approximately 5 minutes. The agarose gelled in about 2 minutes and the stamp was withdrawn with resultant microwell arrays in the agarose gel substrate. After the agarose gelled, arrays were primed by incubation with EB differentiation medium (1:1 mixture IMDM and F-12 Nutrient Mixture (Ham) (Invitrogen), 5% fetal bovine serum (Invitrogen), 1% (vol/vol) insulin transferrin selenium-A supplement (Invitrogen), 55 μM monothioglycerol (Sigma-Aldrich), 100 U L⁻¹ penicillin, and 0.1 mg L⁻¹ streptomycin (Invitrogen) overnight at 37 °C and 5% CO₂.
For hiPSC-EBs formation, 1.2 × 10⁶ dissociated hiPSC in a 50 μl suspension were placed in each microwell array and allowed to sediment into the microwells. After 24-hour incubation at 37 °C, three-dimensional EB were aspirated from the microwells and transferred to a 35 mm tissue culture dish (BD Biosciences). The cells were kept in suspension culture in basal hepatocyte medium under gentle agitation on an orbital shaker at 37 °C and 5% CO₂ with medium changes every other day.

Human neuroblastoma SH-SY5Y cells were grown in Dulbecco’s modified Eagle’s medium supplemented with Nutrient mixture F-12 (Ham) (1:1, v/v) (Gibco, Gaithersburg, MD, USA), 2 g/L sodium bicarbonate (Sigma-Aldrich), 100 units/ml penicillin, 100 μg/ml streptomycin and 10 % fetal bovine serum (FBS). Cells were grown at 37°C in a humidified air with 5% CO₂. SH-SY5Y cells were grown to 80–90 % confluence and then sub-cultured into different culture plates. The cDNAs of WT ALDH2 (GenBank ID: BC002967) and (E504K) ALDH2*2 mutant were constructed and subcloned into a mammalian expression vector pcDNA3 (Invitrogen) containing FLAG-tag sequences (DYKDDDDK). Both plasmids were transfected to SH-SY5Y cells using Lipofectamine 2000 (Invitrogen). Two days after transfection, SH-SY5Y cells stably expressing WT ALDH2 and mutant ALDH2*2 were selected in the presence of 1.5 mg/ml G418 (geneticin sulfate) (Sigma-Aldrich). Positive clones were confirmed by Western blot analysis as described below and maintained in the medium containing 1 mg/ml G418.

ATDC5 mouse cells (Riken Cell Bank, Ibaraki, Japan) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) and Nutrient Mixture F-12 (Ham) (Gibco, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS; Gibco), 100 IU/mL penicillin, and 100 µg/mL streptomycin. ATDC5 cells were induced to chondrogenic differentiation using ITS (insulin (10 μg/mL), transferrin (10 μg/mL), and sodium selenite (3 × 10⁻⁸ M)), as described previously [31 (link),32 (link)]. SW1353 human chondrosarcoma cells (American Type Culture Collection, Manassas, VA, USA) were cultured in DMEM/F-12 media supplemented with 10% FBS, 100 IU/mL penicillin, and 100 µg/mL streptomycin. All cells were maintained at 37 °C in a humidified 5% CO₂ atmosphere.