Dimethyl sulfoxide (dmso)

Hepatocytes were differentiated as described,⁵³ (link) with some modifications. The differentiation process was divided into three stages: pretreatment step, differentiation step and maturation step. Passage 2-Passage 8 UC-MSCs were seeded at 4×10³ cells/cm² in six-well plates in serum-free medium. Culture medium was switched 24 h later to pretreatment medium based on the serum-free medium supplemented with 20 ng/ml epidermal growth factor (Peprotech), 100 ng/ml activin A (Novoprotein) and 10 ng/mL fibroblast growth factor 4 (Peprotech) for 3 days. Thereafter, differentiation was induced by treating UC-MSCs with serum-free medium containing 20 ng/ml hepatocyte growth factor (Peprotech), 10 ng/mL fibroblast growth factor 4, 0.61 g/L nicotinamide (Sigma), 1% dimethyl sulfoxide (MP Biomedicals), and 1% insulin-transferrin-selenium (Gibco) for 10 days. Then cells were incubated with maturation medium containing 20 ng/mL oncostatin M (Peprotech), 1 μmol/L dexamethasone (Sigma), 1% dimethyl sulfoxide and 1% insulin-transferrin-selenium for 15 days. Medium was changed every 3 days.

The human NPC cell lines CNE-2, C666-1, SUNE-1, HONE-1, and 5-8F were acquired from the Shanghai Cell Institute at the Chinese Academy of Sciences. Gibco BRL provided trypsin, fetal bovine serum (FBS), and DMEM medium, while PBS reagents were purchased from Servicebio Technology Co. Dimethyl sulfoxide was sourced from MP Biomedicals, and isopropyl alcohol, methanol, and ethanol were acquired from Fuyu Fine Chemical Co. RIPA lysate was obtained from Beyotime Biotechnology Co., while the Cell Counting Kit-8 (CCK-8) was procured from Dojindo Chemical Research Institute. Lastly, the ECL chemiluminescence reagent was purchased from Thermo Scientific, Inc. siRNAs for p53 and negative control were purchased from Invitrogen. The siRNA sequences of p53 and negative control are list in Table 1.

The cryopreservation of DRG was performed according to a previous study, with slight modifications (Schwarz et al., 2019). T-DRG and C-DRG were collected in cryovials (Thermo Fisher Scientific, Waltham, MA, USA) with 1 mL freezing medium containing 10% dimethyl sulfoxide (MP Biomedicals, Irvine, CA, USA), 30% FBS (Life Technologies, Carlsbad, CA), and 60% DMEM/F12 (Life Technologies). The cryovials were then placed into a freezing container and cooled to –80°C. They were placed into liquid nitrogen after 24 hours (–196°C). After 1 week, the cryovials were thawed at 37°C, and the content of the T-DRG or C-DRG cryovials was transferred to culture plates and washed three times with DMEM/F12 containing 5% FBS. The T-DRG, C-DRGs or hydrogels were transferred to 12-well plates pre-coated with poly-D-lysine, and were then cultured in DMEM/F12 (Life Technologies) supplemented with 5% FBS (Life Technologies), 100 mg/mL penicillin, 100 mg/mL streptomycin (Life Technologies), and 50 ng/mL nerve growth factor 2.5S Native Mouse Protein (Thermo Fisher) at 37°C, 5% CO₂ for 7 days.

To evaluate cell proliferation, an MTT assay was performed. A549 cells were seeded into a 96-well plate at a density of 6×10³ cells/well. At 24 h after seeding, cells were treated with various concentrations of tunicamycin (1.25–10 µg/ml) for 8 h. For combined treatment, cells were treated with 1.25 µg/ml tunicamycin for 8 h followed by 24 h of cisplatin treatment (1.25–40 µg/ml). Cells without drug treatment were used as the negative control. Five wells were examined for each group.
Subsequent to the chemotherapeutic treatments, 20 µl MTT solution (5 mg/ml) was added to each well. Following 4 h of incubation at 37°C, the medium was removed and 200 µl dimethyl sulfoxide (MP Biomedicals, LCC, Santa Ana, CA, USA) was added to each well to resuspend the MTT metabolic product. The absorbance of the dissolved formazan was measured at 492 nm (A492) using a microplate spectrophotometer (Model 500, Bio-Rad Laboratories, Inc., Hercules, CA, USA). The rate of growth inhibition was determined using the following formula: Growth inhibition rate (%) = (1 - [A492_Sample/A492_Control] × 100%. The half maximal inhibitory concentration (IC₅₀) was calculated using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA).

Two glioma cell lines, U251 and SNB19, were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China) and incubated in 5% carbon dioxide at 37 °C. The cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 U/mL penicillin and 100 mg/L streptomycin).
VPA and MHY1485 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide was purchased from MP Biomedicals LLC (Santa Ana, CA, USA). Antibodies, including β-actin, caspase-3, cleaved caspase-3, Bcl-2, Bax, p62, LC3B, AKT, p-AKT, mTOR, and p-mTOR, were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary biotinylated antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).