Cell Biol Toxicol (2011) 27:69–81 77 Fig. 5 Reversibility of fluoride effects. After treatment with fluoride for 24 and 72 h, fluoride was withdrawn by changing the culture medium (Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum), followed by further growth for 24 h. a Cells were fixed and subjected to immuno- cytochemistry for the presence of K10 and K15. Cell nuclei were displayed by DAPI coloration. b RNA was isolated, reversed transcribed, and amplified. Displayed cells had been treated by fluoride for 24 h; cells treated for 72 h gave similar results. K10 and K15 expression appeared reduced even after the withdrawal. c RNA was isolated after 24 and 72 h of fluoride treatment, reversed transcribed, and the HSC70 sequences in the cDNA were amplified. Expression of HSC70 was induced during treatment, albeit after withdrawal of fluoride treatment HSC70 did not changed. K19 expression remained unchanged remained intact, apoptosis features were absent, and the cellular ultrastructure was preserved after 3 weeks of NaF treatment albeit with the appearance of stress- related granules. Cell stress was corroborated by the induction of HSP70 and its deposition in cytoplasmic inclusions. In particular, we show for the first time that the high dose of NaF selectively suppressed t e r m i n a l d iffer e n t i a t io n k e r a t i n s K 1 / 1 0 a n d stratification-related keratin K15, at both mRNA and protein levels. Surprisingly, the effect of NaF at high dose on HaCaT cells seemed to be not reversible. It seems probable that the fluoride effects on keratin gene expression are mediated by the CCAAT/enhanc- er binding protein (c/EBP) family member (Oh and Smart 1998), because this factor appeared reduced by fluoride too. Concerning stratification, we suggest