76 72 h (not shown). The presence of K10 and K15 was assayed by immunocytochemistry, and cell nuclei were displayed by DAPI coloration. It was found that the expression of K10 and K15 was not restored; therefore, the fluoride effect was irreversible under our conditions. Fluoride treatment for 24 and 72 h gave the same results. The expression of HSP70, a cytoplasmic marker of oxidative stress, was increased after high dose of NaF-treated cells. Most HSP70 appeared in granules that increased in number upon fluoride treatment (Fig. 4). The size of the granules was similar to those seen at the EM level in cells treated with the high fluoride dose (see below). This result suggested that NaF at high dose induced cellular stress. It should be noted that indications of apoptosis or necrosis, such as chromatin condensation and fragmentation, vacuoles, and cell fragmentation, were not observed (Figs. 3, 4 and 5). Ultrastructural examination The cells were kept in culture at low or high dose and without NaF for 3 weeks in order to provide information about the stratification capacity and to examine eventual pathological effects of fluoride treatment. For this study, all cultures were supple- mented with 1% FCS. In control cultures (Fig. 6), the cells formed a stratified epithelium made of cubical basal cells and overlaid with two to three polygonal suprabasal cells (Fig. 6a). Desmosomal structures were rather small and few at sites of cell–cell Fig. 4 Analysis of HSP expression. Cells were cultured on cover slips and treated with NaF for 24 h. Immunocytochemistry was performed with HSP70 antibody (clone 5A5 from Abcam), revealed by red fluorescence. Signal appears in cytoplasmic granules at high dose of fluoride treatment Cell Biol Toxicol (2011) 27:69–81 contacts. The keratin intermediate filaments were sparsely scattered in the cytoplasm and associated with the desmosomes at the cell membrane. NaF treatment at either dose did not alter vital structures such as the nucleus, mitochondria, Golgi apparatus, desmosomes, cytoskeletal morphology, or the cell membrane (Fig. 6e, f) when compared to control (Fig. 6d). In the cytoplasm, electron dense bodies were observed resembling myelin figures or stress granules with distinct outlines. The number of these granules increased markedly in the presence of fluoride (Fig. 4). Stratification was unaffected at the low fluoride dose; however, at high dose, the epithelium remained unstratified (Fig. 6c) with flat- tened cells attached together by small desmosomes. Apoptotic features such as chromatin condensation, nuclear fragmentation, or membrane blebbing did not occur at high fluoride dose, neither were necrotic characteristics observed, such as large vacuoles or cell fragments. It was concluded that the high NaF dose exerted some cell stress, but no apoptosis or toxic effects leading to necrosis. Discussion In this study, treatment of HaCaT keratinocytes with high dose of NaF resulted in an inhibition of cell differentiation. The pattern of keratin expression was changed, maintaining a pattern typical for non- stratified cells, and cells failed to undergo stratifica- tion. The proliferation and viability of the cells