Cell Biol Toxicol (2011) 27:69–81 France) and examined with a Leitz DMRB micro- scope equipped with epifluorescence illumination. Ultrastructural analysis The cells were kept in culture with and without NaF at low or high dose for 3 weeks on nitrocellulose mem- branes, in order to allow stratification. All cultures were supplemented with 1% fetal calf serum (FCS). Ultra- structural examination was performed as described previously (Shabana et al. 1994). The cells were washed in sodium cacodylate buffer containing 0.1 M sucrose, pH 7.4, fixed for 30 min in 2% glutaraldehyde in washing buffer, and post-fixed for 30 min in 0.5% osmium tetroxide in cacodylate buffer. After washing and dehydration in increasing concentrations of ethanol solutions and propylene oxide, the samples were embedded in epon-araldite. The blocks were sectioned vertically to examine epithelial stratification. Thin sections were stained with lead citrate for 30 min and examined with a Philips CM 12 electron microscope. Results RNA analysis by RT-PCR HaCaT cells were treated with NaF at low and high dose for 24, 48, and 72 h. RNA was extracted, 73 reverse-transcribed, and specific keratin-coding, c/ EBP alpha-coding, and HSP70-coding segments were amplified under standard conditions. Non-treated cultures (C) were processed in parallel. After ampli- fication, samples were electrophoresed on agarose gels, and the presence of amplified fragments was revealed by ethidium bromide staining of bands of the defined sizes. In the course of the fluoride treatment, the band strengths of K13 and K19 remained the same, as did the actin band (Fig. 1). Bands for K1, K10, and K15 were reduced in the samples subjected to high dose fluoride treatment; no significant change occurred when cells were treated with the low dose at any time period. Treatments for 24, 48, and 72 h reduced K1, K10, and K15 RNA similarly (Fig. 1). We also tested the presence of RNA coding for factors regulating K1 and K10 expression. RNA for c/EBP alpha, an upstream enhancer factor, was markedly reduced by the high dose NaF. c/EBP alpha reduction (Fig. 1) was consistent in the three time intervals, and thus closely correlated with K1 and K10 protein and RNA expression. We also tested the reversibility of the fluoride effect on keratins 10, 15, and 19. After 24 h of withdrawal, bands for K19 were unchanged, whereas K10 and K15 remained at the low level obtained with fluoride treatment (Fig. 5b). HSC70 expression was enhanced after 24 and 72 h of treatment with 5 mM NaF (Fig. 5c). After withdrawal, the HSC70 band Fig. 1 RNA analysis by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was harvested after 24, 48, and 72 h of low (0.5 mM) or high dose (5 mM) NaF treatment as indicated, followed by semiquantitative RT-PCR analysis of RNA. C, cells without fluoride treatment. Actin RNA served as internal control. Keratins 1, 10, 13, 15, and 19 represent the amplified fragments of the different mRNA keratins. K1/10 and K15 detection diminished after high dose of NaF treatment. K13 and K19 remained unchanged at high dose. Transcription factor c/EBP alpha diminished in cells treated at high dose of NaF at the three times of treatment