FIGURE 6.

Reversion of EMT by serum in EFI-hypoxia pre-treated CAL33 cells. (A) Immunofluorescence analysis of vimentin protein expression after 20, 30, and 80 days of culture in EFI-hypoxia (right panel). Pre-treated cells did not form a monolayer with epithelial, cobblestone-like morphology, but they rather displayed a spindle-shaped mesenchymal phenotype. The mesenchymal transformation of cells was confirmed by the progressive invasion of vimentin-positive cells in the successive cultures treated with hypoxia and EFI medium. After 80 days in EFI-hypoxia the cells were transferred to normoxic conditions for 3 or 10 days in the same medium (middle lower panel) or maintained in hypoxia with the addition of 10% serum for 3 or 10 days (middle upper panel). (B) Western blot analysis of vimentin expression in total cell extracts from EFI-hypoxia pre-treated CAL33 cells (80 days) and then cultured for 3, 6, 10 days in the indicated conditions, in normoxia (upper panel) or hypoxia (lower panel). After 10 days in normoxia the protein levels of vimentin were still detectable in CAL33 cells. CAL33 cells: Human tongue squamous cell carcinoma line; EFI: Serum-free medium with EGF, FGF and insulin; EGF: Epidermal growth factor; FGF: Fibroblast growth factor; CAIX: Carbonic anhydrase 9; Ard-1: ADP-ribosylation factor domain protein 1.