TNF enlarges the stem-like cell compartment in human melanomas in vitro . A. TNF decreased the proportion of melanosphere cells resting in the G0 phase of the cell cycle to the level found in adherent monolayer (ML) cultures. TNF (0.5 μg/ml) was added at the time of seeding cells for a sphere-forming assay. After 7 days, melanosphere cells were dissociated, reacted with an anti-Ki67 primary antibody and an anti-mouse Cy5 secondary antibody, and then stained with propidium iodide (PI) before performing the flow cytometry analysis. Ki67-negative cells in the G0/G1 fraction were considered the G0 quiescent cells. B. Flow cytometry of dissociated melanosphere cells revealed that TNF increased the proportion of GFPhigh cells. Representative dot plot data and the corresponding % of GFP-positive cells (left panel) and summary histograms of all data (right panel). C. TNF stimulates stem cell-related sphere-forming abilities in HBL and SK-Mel28 melanoma cell lines. D. TNF increases expression of ABCB5, which is a melanoma stem cell surface marker, in GFPhigh cells (GFPhighABCB5high) when compared to GFPhigh cells in untreated controls (CTR) set at “1” for each cell line. ***p < 0.001; **p < 0.01; *p < 0.05.