Transient exposure to TNF induces irreversible functional changes in the GFPhighstem-like cell compartment. A. Schematic representation of the experimental design. Green nuclei refer to GFP-positive cells. B. TNF affects the pool of self-renewing GFPhigh cells. Tetracycline-induced HBL-H2BGFP melanoma cells formed melanospheres in the presence or absence of TNF that 7 days later were dissociated, and cells were sorted by FACS. The GFPhigh and GFPlow cells were assayed for their sphere- (B) and colony (C)-forming abilities in TNF-free medium. The histograms represent accumulated data from 24 individual samples. C. Representative image of clonogenic assay. The numbers indicate the % of colony-forming units. D. TNF blocks melanoma cell maturation. Representative skin equivalents (SEs) co-cultured with HBL melanoma cells and untreated or treated with TNF (0.5 μg/ml) for 3 weeks with TNF added to fresh medium, which was changed every 3 days. Experiments were repeated 3 times. E. Control (CTR-black) or TNF-treated (TNF-red) dissociated SE cells were evaluated for their ability to generate successive generations of spheres in TNF-free medium. The first (G1), second (G2) and the third (G3) generation spheres were formed during 7 days. ***p < 0.001; **p < 0.01.