TNF expands the subset of GFPhigh/ABCB5highmelanoma stem-like cells through the AKT-signaling pathway. A. Representative image of western blot (n = 2) analysis showing an increase in phosphorylated (p)-AKT in spheres formed by HBL melanoma cells cultured in the presence of TNF (0.5 μg/ml). Actin served as a loading control. B. Representative fluorescent microscopy images showing that LY294002 (10 μm) addition at sphere seeding in the presence or absence of TNF stimulates Melan A expression, and the morphological differentiation of melanospheres was inhibited by TNF. For each condition spheres were collected from 24 wells before dissociation and immunocytochemistry analysis. Scale bar = 20 μm. C. PI3K/AKT inactivation blocked the TNF-induced melanoma SC-related ability to form spheres. Melanospheres were formed in the presence or absence of TNF (0.5 μg/ml) and LY294002 (10 μm). *p < 0.05; ***p < 0.001. The data represent 2 independent experiments in 3 repetitions. D. Flow cytometry data showing that the PI3K/AKT inhibitor LY294002 (10 μm) added at seeding decreased the proportion of TNF-induced ABCB5high cells in melanospheres formed by TNF-treated HBL cells. Note that the NFκB inhibitor BAY 11-7082 (1 μm) did not repress TNF-mediated induction. Data of at least 2 independents each combining spheres from 24 wells.