Sc 63088 5

Small interfering RNA (siRNAs) targeting IL-6 (S7312) and Bcl-X_L (120717) were purchased from Ambion (Austin, TX, USA). siRNAs targeting SOD2 (sc-41655), β-catenin (sc-44275), and STAT3 (sc-29209) as well as lentiviruses expressing small hairpin RNAs (shRNAs) targeting SOD2 (sc-41655-V), Bcl-X_L (sc-43630-V), and SULF2 (sc-63088-V) were obtained from Santa Cruz Biotechnology.

GFP-expressing cells were obtained by transfecting H460 cells with the pEGFP-C1 vector (Clontech, Mountain View, CA, USA) and selecting them in 1 mg/mL of G418 sulfate (Santa Cruz Biotechnology). These transfectants were infected with lentivirus particles expressing control or SULF2-specifc shRNA (Santa Cruz Biotechnology, sc-63088-V), according to the manufacturer's instructions. The infected cells were selected using 1 μg/mL of puromycin (Santa Cruz Biotechnology), and then injected into the mice to form xenograft tumors, as described above. When the tumors reached a volume of ∼500 mm, they were irradiated with 2 Gy of γ-rays. After 3 days, the mice were anesthetized, and blood was obtained through cardiac puncture and mixed with 20-fold higher volumes of RBC-lysis buffer (Intron Biotech, Seoul, Korea). Cells were collected by centrifugation (350 × g, 5 min), resuspended in PBS, stained with DAPI, and analyzed by confocal microscopy. A total of >3,000 stained cells were analyzed per mouse and circulating tumor cells were identified as GFP- and DAPI-positive cells [12 (link), 38 (link)].