Cellular Internalization and DNA Damage

Human normal (TIG-3, MRC5 and WI38) and cancer (breast carcinoma-MCF-7, melanoma-G361 and osteosarcoma-U2OS) cells were obtained from Japanese Collection of Research Bioresources (JCRB, Japan). The cells were authenticated by the source. Cells were frozen in -80 °C and LN₂ in multiple vials and were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY, USA) and treated either with Wi-A or Wi-N at about 60% confluency. Internalization of Wi-A and Wi-N was detected by immunostaining with the anti-WiNA Ab raised in our laboratory. The treated cells were also immuno-stained with a variety of other antibodies that included anti-γH2AX (Millipore #07-627), anti-ATR1 (Abcam, #ab4471), anti-CHK1 (Cell Signaling #2345S), anti-p53 (Santa Cruz. #sc-126) and anti-CARF antibody was raised in our laboratory^{63 (link)}.