Figure 1. Experimental flow.

a. Human foreskin fibroblasts were transfected with lentivirus containing PIWIL2 and GFP, plated on media containing pluromycin plus LIF and cultured for four weeks until the culture was confluent and spheroids developed. b. A single spheroid was harvested, treated with trypsin, and the resultant cells were re-plated. Spheroids that developed after incubation for fourteen days were harvested for further analysis. c. Several spheroids were used for gene expression analysis. d. A single spheroid was used for karyotyping. e. Cells isolated from four spheroids were used for subcutaneous injection into athymic mice (two sites per mouse) for in vivo tumor development. Figure was drawn by Deying Zhang.