Soft Agar and In Vivo Tumorigenicity Assays

Stably miR-31-transfected C666-1 and control cells were subjected to the soft agar assay for anchorage-independent growth in 4 mL medium supplemented with 0.35% agarose and layered on a 5 mL base of 0.7% agarose [41 (link)]. Experiments were carried out in triplicate. After 40 days, cells were stained with 0.8 mM p-iodonitrotetrazolium violet (Sigma-Aldrich). The in vivo tumorigenicity assay was performed as described previously [41 (link)]. A total of 2 × 10⁶ C666-1 cells stably expressing miR-31 or controls were subcutaneously inoculated into the flank of female BALB/c nude mice (nu/nu) (4 mice/construct). Tumor growth was monitored and the tumors were excised at the end of the experiment. All experimental procedures were approved by the Animal Ethics Committee of the Chinese University of Hong Kong.