Penicillin streptomycin p s

HeLa wt, HeLa BAX/BAK KO and BAX/BAK DKO U2OS cells were cultured in low‐glucose Dulbecco’s modified Eagle’s medium (DMEM) (Sigma‐Aldrich), MEF BID KO cells with high‐glucose DMEM and HCT WT, BAX/BAK DKO, BAX/BAK/BID TKO, all BCL‐2 KO and all BCL‐2 KO BOK KO cells with McCoy’s 5A medium (Gibco); all of them supplemented with 10% foetal bovine serum (FBS), 1% penicillin–streptomycin (P/S) (Thermo Fisher Scientific) at 37°C in a humidified incubator containing 5% CO₂. Nalm6, Nalm6 199R, Nalm6 199R BID, BAK and BOK single KO cells were cultured in with RPMI (Gibco) media with 20% foetal bovine serum (FBS), 1% penicillin–streptomycin (P/S) (Thermo Fisher Scientific) and incubated under the same conditions. HCT116 WT, BAX/BAK DKO HCT116 were a generous gift from Dr Schulze‐Osthoff from the University of Tübingen. MEF BID KO and HCT all BCL‐2 KO were generated previously (Andree et al, 2014 (link); O'Neill et al, 2016 (link)). BAX/BAK DKO U2OS ATCC^® HTB‐96. BAX/BAK/BID TKO HCT116, BOK KO all BCL‐2 KO HCT116, Nalm6 199R BID, BAK and BOK single KO cells were generated by CRISPR/Cas9.

Isolated PBMCs were resuspended at a concentration of 10x10⁶ cells/ml in complete medium, consisting of RPMI 1640 supplemented with 2mM L-glutamine, 1% penicillin-streptomycin (P/S) (GIBCO-Thermo Fisher Scientific, Waltham, MA, USA) and 10% autologous human serum from each patient and cultured for 48h in an incubator at 37°C and 5% CO2.
A549 and KU812 cells (Sigma-Aldrich, Saint Louis, MO, USA) were maintained in RPMI-1640, 2mM L-Glutamine, 10% heat-inactivated Fetal Bovine Serum (FBS), 1% penicillin-streptomycin (P/S) (GIBCO-Thermo Fisher Scientific, Waltham, MA, USA) in an incubator at 37°C and 5% CO₂. ATRA (Sigma-Aldrich, Saint Louis, MO, USA), pan-RAR antagonist (AGN 193109), and RARβ antagonist (CD 2665) (Santa Cruz Biotechnology, Dallas, TX, USA) stock solutions were made in DMSO and added to cultures at 1μM final concentration when required. Cell cultures were pre-treated with antagonists for 1h before treatment with ATRA.

We utilized seven cell lines in this study. The cell lines K562, SK-hep-1, HepG2, and CCD18co were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA). Whereas the cell lines Jurkat, U266 and OPM2 were acquired from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany). We cultured K562, U266, Jurkat, and OPM2 in RPMI1640 medium (Pan-Biotech, Aidenbach, Bavaria, Germany) supplemented with 10% FBS (Sigma-Aldrich Chemie GmbH, Munich, Germany) and 1% penicillin/streptomycin (P/S) (Gibco, Schwerte, Germany). While SK-hep-1, HepG2, and CCD18co cells were maintained in EMEM medium (Pan-Biotech, Aidenbach, Bavaria, Germany) supplemented with 10% FBS (Sigma-Aldrich Chemie GmbH, Munich, Germany) and 1% penicillin/streptomycin (P/S) (Gibco, Schwerte, Germany). Meticrane (Sigma-Aldrich Chemie GmbH, Munich, Germany) was dissolved in DMSO and stored at -20°C at a concentration of 200mM. The HDAC inhibitor CUDC-101 (Selleck Chemicals GmbH, Munich, Germany) and the selective HDAC6 inhibitor ACY1215 (Cayman Chemical, Ann Arbor, Michigan, US) was dissolved in DMSO and stored at -20°C at a concentration of 50mM. Also, DNMT1 inhibitor 5-Azacytidine (5AC) (STEMCELL Technologies Germany GmbH, Cologne, Germany) was dissolved in DMSO and stored at -20°C at a concentration of 25mM.

Mouse primary hepatocytes were isolated from the liver tissue of 8-week-old C57/B6 mice by the traditional collagenase perfusion protocol [34 (link)]. Primary hepatocytes, e-iHeps, and r-iHeps were maintained in hepatocyte culture medium (HCM), consisting of DMEM/F-12 (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Seradigm), 0.1 μM dexamethasone (Sigma), 10 mM nicotinamide (Sigma), 1% insulin-transferrin-selenium (ITS) premix (Gibco), 1% penicillin/streptomycin (PS) (Gibco), GlutaMAX™ (Gibco), 10 ng/ml fibroblast growth factor 4 (FGF4) (Peprotech), 10 ng/ml hepatocyte growth factor (HGF) (Peprotech), and 10 ng/ml epidermal growth factor (EGF) (Peprotech). Freshly isolated mouse primary hepatocytes were cultured onto gelatin-coated dish and collected after 48 hrs for RNA extraction. Mouse embryonic fibroblasts (MEFs) were isolated through single-cell dissociation of E13.5 C57/B6 mouse embryos after removing the head and all the internal organs, including the liver and intestine. Isolated MEFs were cultured in MEF medium (MEFM), composed of DMEM high glucose (Biowest), 10% FBS (Seradigm), 1% MEM/NEAA (Gibco), 1% penicillin/streptomycin (PS) (Gibco), and GlutaMAX™ (Gibco).

Human umbilical cord mesenchymal stromal cells (UC-MSCs) were obtained from previous work conducted by Mennan et al. [42 (link)]. Briefly, umbilical cords were sourced from the Robert Jones and Agnes Hunt Orthopaedic Hospital, with informed patient consent (10/H10130/62). Isolation occurred by mincing the tissue before enzymatic digestion with 1 mg/mL collagenase I for 1 h at 37 °C [43 (link)]. Hereafter, UC-MSCs were expanded on tissue culture plastic before further expansion using the Quantum hollow-fibre bioreactor cell expansion system (Terumo UK Ltd., Surrey, UK).
For this study, passage 5 UC-MSCs were cultured on plastic in DMEM-F12 (Fisher Scientific UK Ltd., Loughborough, UK), supplemented with 10% foetal bovine serum (FBS, Fisher Scientific UK Ltd., Loughborough, UK) and 1% penicillin-streptomycin (P/S, Fisher Scientific UK Ltd., Loughborough, UK) in a 37 °C incubator with 5% CO₂. After 48 h, UC-MSCs were washed with PBS (Fisher Scientific UK Ltd., Loughborough, UK) thrice, before exchanging for the four different media compositions (see Table 2).

Sabinene, NAC, and 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI) were purchased from Sigma–Aldrich (St. Louis, MO, USA). DMEM, FBS, horse serum, and phosphate-buffered saline (PBS) were purchased from Hyclone (Logan, UT, USA). Penicillin/streptomycin (P/S) and trypsin-ethylene diamine tetraacetic acid (EDTA) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Antibodies including Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (Life Technology, Carlsbad, CA, USA), anti-p38 MAPK, anti-phospho p38 MAPK, and anti-ERK1/2 (Cell Signaling, Danvers, MA, USA), anti-myosin heavy chain (MYH)-2, anti-phospho ERK1/2, anti-MuRF-1, and anti-β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used in this study.