H1299 cells

H1299 cells (KCLB, Seoul, Republic of Korea, 25803) were acquired from the Korean Cell Line Bank and maintained in RPMI 1640 medium (Gibco, Grand Island, NY, USA). HEK293T cells (CRL-3216) were purchased from the American Type Culture Collection and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, Glendale, AZ, USA). Both cell lines were maintained with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY, USA) and 1× penicillin–streptomycin solution (HyClone, Logan, UT, USA). The cells were cultured at 37 °C in a humidified incubator with 5% CO₂.

Human umbilical vein ECs (HUVECs) and human coronary artery ECs (HCAECs) were purchased from Lonza (Basel, Switzerland), and H1299 cells (NSCLC cell line) were purchased from Korean Cell Line Bank (Seoul, Korea). Cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂. For maintenance, HUVECs and HCAECs were cultured in EGM‐2 medium (Lonza, CC‐3162) supplemented with 1× penicillin–streptomycin (P/S; Thermo Fisher Scientific, Waltham, MA, USA), and H1299 cells were cultured in RPMI1640 (Welgene, Daegu, Korea) supplemented with 1× P/S and 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA). Cells were passaged at approximately 80% confluence with complete growth media.

HEK293T cells, A549 cells, U2OS cells, HeLa cells, and H1299 cells were purchased from Korea Cell Line Bank (KCLB), and MDM2^−/−p53^−/− and MDM2^+/+p53^−/− Mouse Embryonic Fibroblast cell lines were kindly supplied by G. Lozano [42 (link)]. HeLa cells, HEK293T cells, and Mouse Embryonic Fibroblast MDM2^−/−p53^−/− and MDM2^+/+p53^−/− cell lines were grown in 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin, Dulbecco’s Modified Eagle’s Medium (DMEM). U2OS cells, A549 cells, and H1299 cell lines were grown in 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin, Roswell Park Memorial Institute 1640 (RPMI 1640). Cells were grown in 5% CO2 and 95% air at 37 °C.
Either Effectene (Qiagen, Hilden, Germany) or Mirus LT-1 (Mirus, Madison, WI, USA) were used for DNA plasmid transfection following the manufacturer’s instructions in other cell lines. The calcium phosphate/DNA co-precipitation method [43 ] was used for DNA plasmid transfection in the HEK293T cell line. Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) was used for siRNA transfection according to the manufacturer’s instructions.
The control siRNA sequence was 5′-CCUACGCCACCAAUUUCGU-3′. The USP47 siRNA sequences were #1 5′-GTGCAAAGGCCATGAATGA-3′ and #2 5′-TGAAAAGGGATGTGCAAAA-3′’ (Genolution, Seoul, Korea).