Penicillin and streptomycin

THP-1 monocytes were purchased from the German collection of microorganisms and cell cultures (DSMZ). Stable isotope labeling with amino acids in cell culture (SILAC, Ong et al., 2002 (link), 2003 (link)) was achieved by propagating cells in RPMI 1640 free of L-lysine, L-arginine and L-glutamine (PAA Laboratories) supplemented with 220 μM L-lysine and 144 μM L-arginine in either their light (Lys-0 and Arg-0) or heavy isotope-labeled forms (¹³ ${\text{C}}_6^{15}$ N₂-L-lysine/Lys-8 and ¹³ ${\text{C}}_6^{15}$ N₄-L-arginine/Arg-10) (Silantes), 2 mM L-glutamine (Sigma-Aldrich), 10% heat-inactivated dialyzed fetal calf serum (FCS) (Sigma-Aldrich) and the antibiotics penicillin and streptomycin (Biochrom) at concentrations of 100 units/ml and 100 μg/ml respectively. Cell culturing was performed at 37°C and 5% CO₂ in a humidified atmosphere. Cells were grown for at least six cell doublings to ensure complete incorporation of labeled amino acids. For differentiation, phorbol-12-myristate 13-acetate (PMA) (Sigma-Aldrich) was added to a final concentration of 100 nM. After 3 days, the PMA supplemented media was removed, cells were washed with PBS and rested in fresh PMA-free media for further 24 h in order to obtain phenotypic characteristics of macrophages (Daigneault et al., 2010 (link)).

PBMC were isolated using Ficoll–Paque (Pharmacia, Uppsala, Sweden) gradient centrifugation from citrated peripheral blood samples. PBMC were then resuspended in RPMI-1640 medium (Roswell Park Memorial Institute) from Bio-Whittaker, Verviers, Belgium, containing 10 mM of L-glutamine (Biochrom, Berlin, Germany), 0.1 mg/mL of penicillin and streptomycin (Biochrom), and 10% fetal bovine serum (Biochrom) [18 (link)]. The cell concentration was adjusted to 2 × 10⁶ cells/mL. PBMC were stimulated with a pool of spike peptides (PepTivator S, cat. 130-126-701, PepTivator S1, cat. 130-127-048, PepTivator S+, cat. 130-127-312, Miltenyi Biotec, Bergisch Gladbach, Germany) at the recommended concentrations for 16 h (37 °C, 5% of CO₂), while negative controls were treated with the same amount of vehicle used to dissolve the peptide mix [14 (link),15 (link),19 (link)]. After 2 h of stimulation, samples were treated with 6.5 µL of GolgiStop (554724, BD Biosciences, La Jolla, CA, USA). The staining was carried out as already published [11 (link)], using the reagent list reported in Table S2. Samples were finally acquired by flow cytometry (CytoFLEX, Beckman Coulter, Chaska, MN, USA).

Human dermal fibroblasts (HDFs) were isolated from residual skin samples obtained during reconstructive surgery with informed consent of the patient (in agreement with the Helsinki Declaration after approval by the Ethical Committee of the Third Faculty of Medicine) at the Prague Burn Centre (Charles University, Third Faculty of Medicine and University Hospital Kralovske Vinohrady). Briefly, small pieces of split-thickness skin grafts were enzymatically treated with 0.25% trypsin (Sigma-Aldrich, St. Louis, MO, USA) at room temperature for 15 min. to separate the epidermis from the dermis. The dermis was then cut into small pieces that were seeded on cultivation dishes and covered with Dulbecco′s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin, Biochrom, Berlin, Germany) [24 (link)]. After a few days migrating cells were trypsinized and expanded by further culturing at 37 °C and 5% CO₂. Cells at passages 7–8 were used in all experiments.

HepG2 (ATCC HB-8065), L6 myoblasts (ATCC CRL-1458), and 3T3-L1 preadipocytes (ATCC CL-173) were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) (Biochrom GmbH, Berlin, Germany) supplemented with 10% fetal bovine serum (Biochrom GmbH, Germany), and 100 U/mL penicillin and streptomycin (Biochrom GmbH, Berlin, Germany). All cells were routinely cultured in a humidified atmosphere with 5% CO₂ at 37 °C.

Human Burkitt’s lymphoma (BL) cell lines DG-75 and RAJI and diffuse large B-cell lymphoma (DLBCL) cell lines SU-DHL-4 (GCB) and U-2946 (ABC) were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), cultured as recommended by the manufacturer in RPMI 1640 media (Biochrom, Berlin, Germany) supplemented with 10% heat-inactivated FCS (Biochrom, Berlin, Germany) and 100 µg/mL penicillin and streptomycin (Biochrom, Berlin, Germany). Cultured cells were regularly tested for mycoplasma contamination and authenticity (cell surface markers by flow cytometry).
AZD5153 (AZD), I-BET 151 (GSK1210151A) (I-BET) and Entospletinib (GS-9973) (Ento) were obtained from Selleck Chemicals (Absource Diagnostics GmbH, Munich, Germany) and prepared according to the manufacturer’s instructions to a 10 mM stock and stored at −80 °C until ready for use.
Cell lines (3.33 × 10 ⁵ cells/mL) were exposed to AZD, I-BET, Ento or DMSO (control) as mono substance (0.001 µM–10 µM) or in combination for up to 72 h (0.01 µM AZD, 0.1 µM I-BET, 1 µM Ento)

Two different types of the murine fibroblast cell line NIH3T3 were used for the experiments: standard NIH3T3 cells (ATCC-Number: CRL-1658; denoted as: NIH3T3) as a control and genetically modified cells for the expression and secretion of human BDNF. According to the previously described protocol [37 (link)], a tetracycline-regulated plasmid (pLOX) was used as lentiviral vector for the generation of a doxycycline-regulated fibroblast cell line (NIH3T3) secreting BDNF as well as expressing green fluorescent protein (GFP) as a marker (denoted as NIH3T3/BDNF). NIH3T3 and NIH3T3/BDNF fibroblasts were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, FG0445, high glucose; Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (FCS, Biochrom) as well as 1% penicillin and streptomycin (Biochrom). Additionally, 1 μg/mL doxycycline (DOX) was added to the cultures of NIH3T3/BDNF fibroblasts for induction and maintenance of the promotor-regulated gene expression of BDNF.

Self-generated primary equine dermal fibroblasts PriFi1 and PriFi2 and previously isolated primary equine melanoma cells were used for the cell culture experiments. The primary equine melanoma cells MelDuWi belong to the cell culture stock of the Clinic for Horses, University of Veterinary Medicine Hannover, Foundation, Germany, while the primary equine melanoma cells eRGO1 were provided by Dr. Barbara Pratscher, Department for Small Animals and Horses, Vetmeduni Vienna, Austria. PriFi1, PriFi2 and MelDuWi were maintained as monolayers in RPMI1640 cell culture medium with stable glutamine (Biochrom GmbH, Berlin, Germany) supplemented with 15% fetal bovine serum (FBS) superior (Biochrom GmbH) and 1% penicillin and streptomycin (10,000 international units (I.U.)/mL / 10,000 μg/mL, Biochrom GmbH) at 37 °C in a humidified atmosphere with 5% CO₂. Melanoma cells eRGO1 were cultured in Dulbecco’s modified Eagle’s high glucose w/Glutamax (4.5 g/L) cell culture medium (GIBCO-Invitrogen, Thermofisher, Darmstadt, Germany) supplemented with 10% FBS superior (Biochrom GmbH) and 1% Antibiotic-Antimycotic (100x; GIBCO-Invitrogen), containing penicillin (10,000 units/mL), streptomycin (10,000 μg/mL) and amphotericin B (25 μg/mL).