Etoposide

U2OS (Korean Cell Line Bank, RRID:CVCL_0042), HEK293A (Korean Cell Line Bank, RRID:CVCL_6910), A-549 (Korean Cell Line Bank, RRID:CVCL_0023), and MDA-MB-231 (Korean Cell Line Bank, RRID:CVCL_0062) cells were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, UT, USA) that is supplemented with 10% fetal bovine serum (FBS, HyClone, UT, USA) and 1% penicillin G and streptomycin (Welgene, Daegu, Republic of Korea). The cells were cultured in a humidified atmosphere that contained 5% CO₂ at 37 °C. None of our cell lines were listed as commonly misidentified cell lines by the International Cell Line Authentication Committee (ICLAC). Prior to the experiments, they were also authenticated through PCR-based short tandem repeat assays. Mycoplasma contaminations were confirmed using MycoAlert® Mycoplasma Detection Kits (Lonza, Basel, Switzerland). U2OS cells were treated with 50 μM of sodium arsenite (SA, Sigma–Aldrich, California, USA) from 2 h to 12 h to induce stress depending on the experiments. U2OS cells were also treated with 50 μM of cisplatin (Sigma–Aldrich, California, USA) and 25 μM of etoposide (Sigma–Aldrich, California, USA) from 2 h to 12 h depending on the experiments.
To generate U2OS cell line that stably expresses VRK2, U2OS cells were transfected with either pNTAP-Mock or pNTAP-VRK2 plasmids using Neon Transfection System (Invitrogen, California, USA). After 24 h of incubation, transfected cells were selected through the treatment with G418 disulfate salt (800 μg/ml, Sigma–Aldrich, California, USA). G418 disulfate salt containing media were changed every 2 days. The overexpression of VRK2 was checked by Western blot.

Human monocytic THP-1 cells were maintained in culture in Roswell Park Memorial Institute medium (RPMI 1640, Invitrogen) culture medium containing 10 % of heat inactivated fetal bovine serum (Invitrogen) and supplemented with 10 mM Hepes (Gibco, #15630-056), 1 mM pyruvate (Gibco, #11360-039), 2.5 g/l D-glucose (Merck) and 50 pM ß-mercaptoethanol (Gibco; 31350–010). THP-1 monocytes are differentiated into macrophages by 24 h incubation with 150 nM phorbol 12-myristate 13-acetate (PMA, Sigma, P8139) followed by 24 h incubation in RPMI medium. Macrophages were polarized in M1 macrophages by incubation with 20 ng/ml of IFN-γ (R&D system, #285-IF) and 10 pg/ml of LPS (Sigma, #8630). Macrophage M2 polarization was obtained by incubation with 20 ng/ml of interleukin 4 (R&D Systems, #204-IL) and 20 ng/ml of interleukin 13 (R&D Systems, #213-ILB). HepG2 and A549 cells were respectively cultivated in Dulbecco’s modified Eagle's minimal essential medium (DMEM medium 1 g glucose/l) (Gibco) and Minimum Essential Medium Eagle medium (MEM) (Gibco), both containing 10 % fetal bovine serum. In the co-culture experiments, THP-1 monocytes were differentiated in 6 Transwell inserts (membrane pore size of 0.4 μm, Corning, #3450). Macrophages and HepG2 cells were co-cultured in CO₂ independent medium supplemented with 0.5 mM L-glutamine (Sigma, # G3126) and 3.75 g/l of D-glucose (Sigma, #50-99-7) for 16 h before being incubated with or without 50 μM etoposide (Sigma, #E1383) for 24 h. Macrophages and A549 cells were co-cultured in CO₂ independent medium supplemented with 0.5 mM L-glutamine and 2.5 g/l of D-glucose for 24 h before being incubated with or without 50 μM etoposide for 16 h. In the monoculture experiments, 0.8 x 10⁶ THP-1 monocytes were differentiated and polarized in 6 well plates. Next, they were incubated in CO₂ independent medium supplemented with 0.5 mM L-glutamine (Sigma, # G3126) and 3.75 g/l of D-glucose (Sigma, #50-99-7) for 16 h before being incubated with or without 50 μM etoposide (Sigma, #E1383) for 24 h.