Advanced dmem medium

Carboxylated polystyrene beads (FluoSpheres^®) were purchased from Molecular Probes™: 40 nm (8795), 100 nm (F8800), 200 nm (F8809). Dyes for Müller cell and viability staining were obtained from Invitrogen: Hoechst 33342 (H3570), FM^® 1-43 (T3163), Mitotracker^® Deep Red (M22426), Propidium iodide (P3566). Antibodies against glutamine synthetase (ab73593) and Collagen IV (ab6586) were purchased from Abcam; AlexaFluor^® 647 tagged secondary antibody (A27040) was obtained from Invitrogen. Cell culture materials were mostly acquired from Gibco™: CO₂ Independent medium (18045088), Neurobasal^®-A medium (10888022), Advanced DMEM medium (12491023), B-27^® supplement (17504044), Penicillin–streptomycin (15140122), l-Glutamine (25030081), Trypsin–EDTA 0.25% (25200072).

The L3.6pl human pancreatic cancer cells were isolated and established after several cycles of in vivo selection in nude mice [58 (link)]. TBO368 was obtained with written consent from a pancreatic cancer patient and subsequently established as low-passage primary cell cultures. The study has been approved by the Ethics Committee of the University of Cologne (BIOMASOTA, (Biologische Material Sammlung zur Optimierung Therapeutischer Ansätze), ID: 13-091, approval in May 2016). L3.6pl was cultured in DMEM medium (Gibco, ThermoFisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (vol/vol), 1% vitamin mixture, 1% sodium pyruvate, 1% nonessential amino acids, 1% L-glutamine and 1% penicillin/streptomycin (FBS from Capricorn Scientific, Ebsdorfergrund, Germany)(others from Invitrogen, ThermoFisher Scientific, Waltham, MA, USA). TBO368 was cultured in advanced DMEM medium (Gibco) supplemented with 10% FBS, 1% L-glutamine and 1% penicillin/streptomycin. Human NK cell line NK92 cells were obtained from the ATCC and cultured in MEM α, no nucleosides medium (Gibco) containing 12.5% FBS, 12.5% horse serum (Gibco), 100 IU/mL penicillin, 100 μg/mL streptomycin, 0.02 mM folic acid, 0.1 mM 2-mercaptoethanol, 0.2 mM Myo-inositol, 2 mM l-glutamine, and 100 U/mL recombinant human IL-2 (Peprotech, Hamburg, Germany).

Porcine mesenchymal stem cells were isolated from adipose tissue and bone marrow from a 3-month-old Polish Large White pig, as described by Kociucka et al. (2016 (link)). The cells were cultured in advanced DMEM medium (Gibco) supplemented with 10% FBS (Sigma-Aldrich), 5 ng/ml FGF-2 (PromoKine), 2 mM L-Glutamine (PAA), 1 mM 2-mercaptoethanol (Sigma-Aldrich), 1 × antibiotic antimycotic solution (Sigma Aldrich), and MEM NEAA (Thermo Fisher Scientific) at 37 °C with 5% CO₂ supplementation. To induce adipogenic differentiation, the cells were grown to confluency and were cultured with adipogenic differentiation medium composed of advanced DMEM (Gibco) with 10% (v/v) FBS (Sigma), 1 × antibiotic antimycotic solution (Sigma Aldrich), MEM NEAA (Thermo Fisher), 5 ng FGF-2 (PromoKine), 1 × linoleic acid albumin (Sigma-Aldrich), 1 × ITS (Sigma-Aldrich), 1 μM dexamethasone (Sigma-Aldrich), 100 μM indomethacin (Sigma-Aldrich), and 50 mM IBMX (Sigma-Aldrich). The differentiation process was allowed to proceed for 7 days and lipid droplet formation was examined using a phase-contrast microscope (TS100 Eclipse, Nikon).

Constructs for stable constitutive expression of TAp63α, TAp63γ, ∆Np63α and ∆Np63γ were provided by Maranke Koster (University of Colorado, Denver, USA) and were cloned using the pQCXIH vector. Retroviral particles containing the described constructs were produced in HEK293-T cells according to a published method [18 (link)]. Briefly, HEK293-T cells were cultured in Advanced D-MEM medium (GIBCO) supplemented with 2% fetal calf serum and a culture additive containing 0.01 mM cholesterol (Sigma-Aldrich), 0.01 mM egg yolk lecithin (Serva Electrophoresis GmbH, Heidelberg, Germany) and 1x chemically defined lipid concentrate (GIBCO) (transfection medium). The cells were co-transfected using the calcium phosphate method with the following three plasmids: a retroviral expression vector together with the two helper plasmids pVSV-G (Clontech), encoding the G-glycoprotein of the vesicular stomatitis virus, and pHit60 encoding the retroviral gag and pol genes (provided by Dr. Christian Buchholz, Paul-Ehrlich- Institute, Langen, Germany). Fourteen hours after transfection the medium was replaced with fresh transfection medium. The supernatant containing each recombinant retrovirus was collected 48 h after transfection, filtered through a 0.45 μm syringe filter and stored in aliquots at - 80°C.

The C2C12 mouse myoblast cell line (American Type Culture Collection, ATCC, Manassas, VA, USA; ATCC^® CRL-1772™) and the L929 mouse fibroblast cell line (Merck, Kenilworth, NJ, USA) were cultured in advanced DMEM medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), supplemented with 5% fetal bovine serum (FBS, Gibco), GlutaMAX (100×, Gibco), and penicillin–streptomycin (100×, Sigma-Aldrich, Merck, Darmstadt, Germany) grown in a 5% CO₂ humidified atmosphere at 37 °C. Cells were free of mycoplasma infection, as confirmed by routine testing with a MycoAlertTM PLUS mycoplasma detection kit (Lonza, Basel, Switzerland).

DRG cultures were obtained as described (Gandini et al., 2014 (link)). In brief, DRG were dissected from C57BL/6 mice (P6-P10) in Advanced DMEM Medium (Gibco) supplemented with 20% of Fetal Bovine Serum (Gibco), washed, and digested for 40 min at 37°C with a mixture of trypsin type XI (1.25 mg/ml, Sigma) and collagenase IV (1.25 mg/ml, Sigma), followed by mechanical dissociation. Cells were spun down at 1,000 g for 5 min at 10°C and re-suspended in Advanced DMEM Medium supplemented with 10% FBS. Cells were plated onto L-lysine-covered coverslips (12 mm, Carolina Biological Supply, Burlington, NC, USA) and kept in a 5% CO₂ humidified atmosphere at 37°C. The Patch-clamp recordings were made 24 h after dissociation (1 day in vitro, 1 DIV).