74 decreased to the level of control cells. Thus, the fluoride effect was reversible for HSC70 expression, but not for that of keratins 10 and 15. Analysis of expression of keratins by Western blots Cells from cultures as in Fig. 1 were harvested, and total protein was extracted. Equal protein amounts were loaded on SDS polyacrylamide gels and electro- phoresed. Proteins were transferred to membranes, and specific keratins were revealed by an antibody reaction followed by luminescence detection (Fig. 2). Presence of actin was tested as an internal loading control; its band strength was unaffected by fluoride. Also, the band strengths for K13 and K19 were similar in all samples, suggesting that fluoride did not influence their synthesis. Bands for K1, K10, and K15 became fainter or invisible in cells treated with the high fluoride dose. The low dose did not change expression of these proteins. Treatment for 24, 48, and 72 h gave similar results (Fig. 2). Immunolabeling of cell cultures Semiconfluent cultures were treated with fluoride for 1, 2, and 3 days and labeled by antisera as indicated. Cell Biol Toxicol (2011) 27:69–81 Similar results were obtained at the three time intervals; only the 24-h results are shown (Fig. 3). Anti-keratin antibodies stained the cytoplasm of HaCaT cells. In parallel, DAPI was added in order to stain cell nuclei. Any view field in the microscope was photographed twice: one time for the blue DAPI fluorescence and one time for the red fluorescence, indicating presence of the specific keratin. For K19, antibody and DAPI labeling detected the same pattern in the cell lawn at both fluoride concentrations and the control. Anti-K13 serum detected frequent cell clusters, indicating expression of K13 in neighbor- hoods of a few cells. Cells outside of these neighbor- hoods did not display K13. Addition of fluoride at low or high dose did not change this pattern. With anti-K1/K10, clustering of positive cells was detected too. Here, the high fluoride dose suppressed expres- sion, whereas the low dose had no effect (b in Fig. 3). K15 was expressed in the control cultures in 30% of the population in an even distribution (m in Fig. 3). The low dose of fluoride provoked an uneven suppression of K15 resulting in a patchy labeling (n in Fig. 3); the high fluoride dose suppressed all K15 (o in Fig. 3). Reversibility was assessed during withdrawal for 24 h, following NaF treatment for 24 (Fig. 5a) and Fig. 2 Analysis of expression of keratins by Western blot. Total protein was isolated from HaCaT cells after treatment with low (0.5 mM) or high dose (5 mM) of NaF during 24, 48, and 72 h and subjected to Western blotting. C, non-treated cells. Monoclonal antibodies LH1, 1C7, LHK 15, and b 170, respectively, against keratins 1/10, 13, 15, and 19 reacted with the expected bands at 56, 52, 62, 57, and 40 kDa, respectively. Anti-actin serum was added as control, eliciting a band at 40 kDa. Keratins 1/10 (K1/10) and 15 (K15) proteins were not detected after high dose of NaF treatment during the three times of observation. Keratin 13 (K13) and keratin 19 (K19) detection was not modified at any exposure time neither at low dose and high dose of NaF treatment