Fig 2: IGF-1 downregulation decreases the expression of cancer stem-like and pluripotency markers in B16-F10 melanoma cells(A) Flow cytometry analysis of the canonical self-renewal transcription factors Oct-3/4 and SOX2 in the C10 clone, B16-F10CT and B16-F10WT cells. Top, Dark-colored bars correspond to cells incubated with the antigen-specific antibody, and gray bars correspond to cells incubated with the isotype-matched control antibody. Mean fluorescence intensity values for each marker are shown at the top left of each histogram. All panels represent at least four independent experiments showing similar results. Bottom, Data are represented as means ± SEM for three independent determinations, p < 0.05, p < 0.01 versus B16-F10CT cells. (B) Western-blot analysis for MITF in C10, B16-F10CT and B16-F10WT cell lysates. β-actin was used as the loading control. Bottom, Graphs show the quantification of three immunoblot signals corresponding to the protein of interest/β-actin ratio, p < 0.01 versus B16-F10CT cells. (C) Analysis of MITF expression (green) by fluorescence microscopy. Blue, nuclei stained with DAPI. The scale bar represents 10 μm. The panel depicts one of three independent experiments. (D) CD24 and CD133 cell surface markers were analyzed by flow cytometry. Top, Panels represent at least four independent experiments with similar results. The percentages of CD24- and CD133-positive cells are shown at the top right of each histogram. Bottom, Data are represented as means ± SEM for three independent determinations, p < 0.05, p < 0.001 versus B16-F10CT cells.