FIG. 2.

Identification of Rh123^low quiescent cells with stem-like properties. (A) After dissociation, OCR_OCMM1 and OCR_OCMM2 spheroid cells were incubated with 0.1 μg/mL Rh123 for 20 min (left panel). After 60 min of Rh123 exclusion, a subpopulation of low Rh123-retaining (Rh123^low) cells appeared (right panel). (B) Rh123^low and Rh123^high cells were sorted by FACS, and the cell cycle was assessed by flow cytometry after KI67/PI staining. A fraction of cells in the G0 phase of the cell cycle is shown in red. *P ≤ 0.05 and ***P ≤ 0.005. OCR_OCMM1 (C) and OCR_OCMM2 (D) Rh123^low and Rh123^high fractions were sorted by FACS, replated at a clonal density of 1,000 cells/mL, and grown under sphere-forming conditions. After 7 days of culture, large tumor-like spheres formed, mainly from the Rh123^low fraction (quantitative graph below). The self-renewal capacity of melanospheres was assessed by dissociating and replating the cells at a clonal density for three successive generations (G₁–G₃). **P ≤ 0.01; ***P ≤ 0.005; and ^###P ≤ 0.005 (* and # for the comparison of Rh123^low vs. Rh123^high and G_n Rh123^low vs. G_n+1 Rh123^low, respectively). FACS, fluorescence-activated cell sorting; PI, propidium iodide; Rh123, rhodamine 123; SFU, sphere-forming unit.