2.3. Cell Culture

We transfected B16F10 cells (1 × 10⁵ cells/well) by using Lipofectamine RNAiMAX (Invitrogen, Waltham, MA, USA). B16F10 wild-type, LRP1 or p53 overexpressing (OE) or knockdown (KD) cells, SK-MEL-28, or A431 cells (1 × 10⁵ cells/well) were seeded in triplicate in 6-well plates (Thermo Fisher Scientific, Lafayette, CO, USA) and were left for 16 h overnight to attach before the treatment started. We treated cells with/without YO-2, or DMSO/PBS controls for 24 h. In some experiments, B16F10 cells were cultured with/without rec. tPA (1500 U/mL) and rec. plasminogen (100 ng/mL). Trypan blue negative cells were determined as viable cells (cat. 207-17081; Fuji Film Wako). We used the LDH assay kit (ab65393, Abcam, Cambridge, UK) to determine cytotoxicity and the Caspase 3/7 activity assay (Promega, Tokyo, Japan) to assess caspase 3 and 7 activity as recommended by the manufacturers.

Cell lines were cultured for 16 h. Then, cells were exposed to UV at 254 nm using UV lamp VL-6.LC (Vilber Lourmat, Eberhardzell, Germany).