Minimum essential medium (mem)

Human neuroblastoma SH-SY5Y cells, hepatoblastoma HepG2 cells, murine macrophage-like RAW 264.7 cells, and fibroblast-like MRC5 cells from normal lung tissue were all purchased from the American Type Culture Collection (ATCC, VA, United States). HepG2, SH-SY5Y, and MRC5 were cultured in Minimum Essential Medium (MEM; Biosera, France) with 5 mmol/L glucose, and supplemented with both 2 mmol/L glutamine, 10% fetal bovine serum (FBS; Biosera), as well as with 1% non-essential amino acids (Biosera) in a normoxic CO₂ chamber at 37°C in a humidified atmosphere. RAW 264.7 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM with 4,500 mg/L glucose, L-glutamine, and sodium bicarbonate) with 10% FBS.
The medium was replaced 24–48 h prior, starting the experiments with fresh medium containing LR and BR in a final concentration of 5, 25, or 50 μmol/L (control samples were treated by medium containing an adequate volume of LR/BR solvents; final concentration of DMSO = 0.5% v/v, concentration of BSA in FBS = 2.5 g/L (Soutar et al., 2019 (link))). Thus, the respective concentrations of Bf (Bilirubin free, unbound, biologically active fraction of bilirubin) corresponded to non-toxic, borderline toxic and toxic concentrations, respectively (Roca et al., 2006 (link); Zelenka et al., 2012 (link)). On the day of the experiment, the control cell culture reached a confluence of 80–90%.

NCI-H520 and HEp-2 cell lines (ATCC) were cultured in RPMI-1640 and MEM, respectively (Biosera, Boussens, France), supplemented with 10% fetal bovine serum (FBS, Biosera) and 1% antibiotics (penicillin/streptomycin; Biosera), and were incubated at 37 °C in a 5% CO₂ atmosphere. Cells were transfected with shRNAs against deadenylases control, using cationic liposomes (Xfect; Clontech, Mountain View, CA, USA), as previously described. All shRNAs were from Sigma-Aldrich (MISSION^® shRNA; Merck KGaA, Darmstadt, Germany). The following shRNA plasmids were used to silence the respective deadenylases: PARN (NM_002582), CNOT6 (NM_15455), CNOT6L (NM_012118), CNOT7 (NM_013354), CNOT8 (NM_004779), NOCTURNIN (NM_012118), and non-targeting control (SHC016). Twelve hours later, cells were selected with puromycin (6 μg/mL). All cell lines used in the study tested negative for mycoplasma.