First, 3 105 (HepG2) or 4 105 (HepaRG) cells were centrifuged at 300 g for 5 min. from APAP-induced cell death. However, the known specific inhibitors of necroptosis (necrostatin-1 and MDIVI) were only effective in differentiated HepaRG, which suggest a differential execution of activated pathways in the two models. By applying 3D culture methods, CYP2E1 mRNA levels could be elevated, but we failed to achieve a significant increase in hepatocyte function; hence, the 3D cultivation especially in APAP toxicity studies is not necessarily worth the complicated maintenance. Based on our findings, the hepatocyte functions of HepaRG may stand between the properties of HepG2 cells and main hepatocytes (PHHs). However, it should be noted that in contrast to PHHs having many limitations, HepaRG cells are relatively immortal, having a stable phenotype and CYP450 expression. for 15 min at 4 C. The supernatant was utilized for protein analysis and stored CHR2797 (Tosedostat) at ?80 C. Protein samples were quantified using the Pierce? BCA Protein Assay Kit (Thermo Scientific?) according to the manufacturers guidelines. 2.9. Western Blot SDS-PAGE was carried out by using Cleaver Scientific (Rugby, UK) omniPAGE system. Proteins were transferred onto Millipore 0.45 m nitrocellulose membrane. Immunoblotting was performed using TBS Tween (0.1%), containing 5% non-fat dry milk for blocking membrane and 1% non-fat dry milk for antibody solutions. Loading was controlled by developing membranes for -actin or GAPDH. The following antibodies were applied: Rabbit PolyAb Anti-PARPI (Proteintech?, Rosemont, IL, USA, 13371-1-AP), Rabbit PolyAb Anti-RIPK1 (Proteintech?, 17519-1-AP), Rabbit PolyAb Anti-RIPK3 (Proteintech?, 17563-1-AP), Anti-P-c-Jun (Cell Signaling, Danvers, MA, USA, 9261S), Anti-c-Jun (Cell Signaling, 9165S), and Anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX, USA, 6C5). Rabbit PolyAb Anti-ACTB (Proteintech?, 20536-1-AP), antiHRP-conjugated secondary antibodies: HRP-Goat Anti-Rabbit IgG (Proteintech?, 00001-2), HRP-linked Anti-Mouse IgG (Proteintech?, 7076S). The bands were visualized using a chemiluminescence detection kit (Thermo Scientific?, 32,106) and VWR? (Radnor, PA, USA) Imager Chemi Premium gel documentation system with VWR? Image Capture Software (version: 1.6.1.0). For densitometry analysis, Western blot data were acquired using ImageJ software bundled with 64-bit Java 1.8.0_172. 2.10. Determination of Caspase-3/7 Activation Cells were treated and prepared as explained above. First, 3 105 (HepG2) or 4 105 (HepaRG) cells were centrifuged at 300 g for 5 min. Cells were resuspended in 50 L of assay buffer (20 mM HEPES, pH 7.4, with 1% CHAPS, 5 mM DTT, and 2 mM EDTA) and stored at ?80 C for 2C3 days. After thawing, the lysates were supplemented with 17 nM Ac-DEVD-AMC (a fluorogenic substrate of caspase-3/7 proteases). The combination was incubated at 37 C for 1 h, and the fluorescence was determined by a fluorescent plate reader (Varioskan LUX, excitation: 380 nm, emission: 445 nm). CHR2797 (Tosedostat) The results were normalized to the protein content of the sample that was determined by Thermo Scientific? CHR2797 (Tosedostat) Pierce? BCA Protein Assay Kit, according to the manufacturers instructions. 2.11. GSH Measurement For the determination of cellular GSH, monochlorbimane (mClB) derivatization followed by HPLC separation and fluorescent detection was used [37,38]. First, 105 trypsinized cells in HBSS (Hanks Balanced Salt Answer, Sigma-Aldrich?) were diluted in Tris buffer (20 mM, pH 8.0) up to 100 ul, which was supplemented with 1 U/mL glutathione-S-transferase enzyme (GST) and mClB to reach 1 mM final concentration. After a 15 min incubation HUP2 in the dark at RT, the derivatization was halted with the addition of 100% trichloroacetic acid (TCA). The solution was centrifuged at 15,000 for 10 min, and the supernatant was utilized for GSH determination. For separation, a Waters Acquity UPLC H-Class system was used, equipped with an Acquity UPLC BEH C18 2.1 50 mm column with an average particle diameter of 1 1.7 m. Gradient elution was used as 0.25% sodium-acetate.