Rpmi 1640 medium

A scanning multi-well spectrophotometer (Multiskan spectrum, Thermo Fisher Scientific, Waltham, MA, USA) was utilized to measure absorbances. RPMI 1640 medium, fetal bovine serum (FBS), penicillin, streptomycin and phosphate buffered saline (PBS) were purchased from Biowest (Nuaillé, France). Lipopolysaccharide (LPS) (from E. coli 0111:B4) and phorbol 12-myristate-13-acetate (PMA) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The human osteosarcoma cell line (U2OS), and the small cell lung cancer (SCLC) cell line (DMS114) were obtained from the American Type Culture Collection (ATCC). The non-small lung cancer cell line (HCC15) was supplied by the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (DSMZ). U2OS cell lines stably transfected with pcDNA3.1 vector containing the sequence encoding the full-length FGFR1 (U2OSR1) or empty pcDNA3.1 vector (U2OS) were prepared as described previously (16 (link)). The U2OS cell line stably transfected with FGFR1-IIIc_K514R (U2OSR1-K514R) was kindly provided by Dr. Ellen M. Haugsten from the Department of Molecular Cell Biology, Institute for Cancer Research (Oslo University Hospital). U2OS, U2OSR1, and U2OSR1-K514R cells were cultured in DMEM (Biowest) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and antibiotics (100 U/ml penicillin, 100 μg/ml streptomycin and 1 mg/ml geneticin). DMS114 cells grew in Waymouth’s MB 752/1 medium (ATCC) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin). HCC15 cells were cultured in RPMI 1640 Medium (Biowest) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific) and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin). All cancer cell lines were kept at 37 °C in a 5% CO₂ incubator.

Mycoplasma-free A549 (human lung adenocarcinoma cell line) and Huh7 (human liver carcinoma cell line) cells were purchased from the Korean Cell Line Bank and maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Biowest, Riverside, MO, USA) and antibiotic-antimycotic solution (Welgene, Seoul, Korea).

CHO K-1 cells overexpressing hPD-L1 and the recombinant TCR ligand (hPD-L1 Antigen Presenting Cells, hPD-L1 aAPCs) (Promega, Madison, WI, USA) and Jurkat T cells overexpressing hPD-1 and carrying a luciferase reporter gene under the control of Nuclear Factor of Activated T-cells Response Element (NFAT-RE) (hPD-1 Effector Cells, hPD-1 ECs, Promega) were cultured in RPMI-1640 medium (Biowest, Billerica, MA, USA) supplemented with 10% Fetal Bovine Serum (FBS, Biowest) and 200 mM L- Glutamine (Biowest) in the presence of G418 (250 µg/mL, InvivoGen, San Diego, CA, USA) and Hygromycin B Gold (50 µg/mL, InvivoGen) as selection antibiotics. The overexpression of hPD-L1 and TCR ligand in aAPCs and PD-1 in ECs were confirmed by flow cytometry and western blot analysis, respectively. PCR tests for Mycoplasma sp. contamination [50 (link)] were routinely performed and indicated negative results for both cell lines.

HL60 and Jurkat cells were acquired from the local cell bank at the Institute of Immunology and Experimental Therapy in Wrocław (Poland), while KG1 cells were purchased from the German Resource Center for Biological Material (DSMZ GmbH, Braunschweig, Germany). The cells were cultured in RPMI-1640 medium (Biowest, Nuaillé, France) with 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin (all from Sigma-Aldrich) and maintained at standard cell culture conditions. DNA from AML patients’ blasts were kept frozen at −80 °C from the study published before [18 (link)].
In order to study the influence of hypomethylating agents, HL60 cells were exposed to 10 nM 1,25D, 1 μM ATRA, 50 or 100 μM zebularine (ZEB), and/or 5 or 10 μM 5-azacytidine (5AZA) for 96 h. The expression of cell surface markers of differentiation was determined by flow cytometry. Cells were washed and stained with antibodies: CD11b-FITC and CD14-PE (ImmunoTools) for 1 h on ice. Next, they were washed with ice-cold PBS and suspended in 0.5 mL of PBS supplemented with 0.1% bovine serum albumin (BSA) prior to analysis on Accuri C6 (Becton–Dickinson). Data analysis was performed using FCS Express (De Novo™ Software, Pasadena, CA, USA).

All human myeloma cell lines (HMCLs) used in this study were grown in RPMI-1640 medium (BioWest) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin and kept at 37 °C with 5% CO₂. The identity of HMCLs was recently authenticated by short tandem repeat analysis. Plasma cells were isolated by CD138 immunomagnetic bead selection from bone marrow aspirates of newly diagnosed patients with MM, which were obtained from the National University Hospital after approval by the institutional ethical committee. Reagents and kits included: DMSO was purchased from Sigma. THZ1 (1604810-83-4) was obtained from Cayman Chemical. JQ1 (SML0974) was purchased from Sigma–Aldrich. Antibodies used are shown in Supplemental information.