4 phenylbutyric acid 4 pba

4-phenylbutyric acid (4-PBA) was supplied from Santa Cruz Biotechnology (Dallas, TX, USA, Cat. sc-232961); 4-(4-nitrophenyl)butyric acid (NPBA, Sigma-Aldrich, Milan, Italy; Cat. N20506, internal standard, IS), thapsigargin (Sigma-Aldrich; Cat. T9033), LC-HRMS grade formic acid, methanol, and water were purchased from Carlo Erba (Milan, Italy, Cat. 414831 and 412111). DMEM and NBA matrices were obtained from Sigma-Aldrich and Thermo Fisher Scientific (Waltham, MA, USA), respectively.

Cells were cultivated under standard conditions (37 °C, 5% CO₂) and grown in melanoma isolation media (MIM): MCDB153 medium / Leibovitz’s L-15 medium (4/1) supplemented with 2% FBS, 7.5% NaHCO₃, 5 µg/ml Insulin, 5 ng/ml EGF, 1.68 mM CaCl₂, 50 mg/L streptomycin sulphate and 30 mg/L penicillin. Cells were split when 90% confluent using a 0.25% trypsin/EDTA solution. If not otherwise indicated, experiments were performed in MIM for 48 hours. 4-phenylbutyric acid (4-PBA) was obtained from Santa Cruz Biotechnology (Dallas, USA) and PD166866 and thapsigargin were obtained from Sigma-Aldrich (St. Louis, USA).

The INS-1E cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 11 mM glucose supplemented with 10 mM HEPES (pH 7.3), 10% heat-inactivated fetal bovine serum (FBS; Invitrogen), 50 μM β-mercaptoethanol, 1 mM sodium pyruvate, 50 μg/mL penicillin, and 100 μg/mL streptomycin at 37 °C with 5% CO₂ in a humidified incubator. ER stresses are induced by thapsigargin (1 μM; Calbiochem, San Diego, MO, USA) for 6 h or tunicamycin (2 μg/mL; Calbiochem) for 16 h treatments with/without 4-phenylbutyric acid (4-PBA, 20 μM; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Oxidative stresses are induced by serum deprivation, nitric oxide (2 mM; Santa Cruz Biotechnology), or hydrogen peroxide (200 μM; Santa Cruz Biotechnology) for 24 h treatments.