Figure 1. Ammonia tolerance enhances putative tumor initiation.

(A) Correlation plot of the GI₅₀ of NH₄Cl versus the number of colonies in soft agar for 15 OVC cell lines. r; Pearson correlation coefficient. (B) CD90⁺ and CD90⁻ subpopulations of PEO1 cells were isolated by cell-sorting and established. Characteristics of cells were assessed in vitro by soft agar assays with an initial seeding number of 2,000 (bar chart, upper panel) and in vivo by serial dilution xenograft assays (table, lower panel) that show the incidence of tumors out of the total number of injection sites. (C) In vivo orthotopic xenograft of CD90⁻ (2 mice) or CD90⁺ (3 mice) PEO1 cells infected with a luciferase-expressing vector. 2 × 10⁶ cells were injected into the peritoneal cavity. The signals from the engrafted cells were detected with IVIS imaging system 4 weeks after injection (Left). A.U., arbitrary units. Right panel shows average intensities of luciferase bioluminescence. (D) Determination of the GI₅₀ of NH₄Cl for the parental cell line (PEO1), CD90⁻ and CD90⁺ subpopulations. (E) Apoptotic and live cell population were determined using Annexin-V-Alexa 647 and Sytox-blue staining in CD90⁻ and CD90⁺ cells after 10 mM NH₄Cl treatment for the indicated time. Error bars indicate s.e.m. ^*P < 0.05; ^**P < 0.01; ^***P < 0.001 (Student’s t-test).