As a control, the cells Selleckchem RG7422 were suspended in 1 mM Tris–HCl (pH 7.2) at a low cell density (2000 cells mL−1) so that encystment could be avoided as much as possible. Aprotinin was purchased from Sigma-Aldrich, leupeptin and pepstatin from Peptide Institute Inc., phenylmethylsulfonyl fluoride (PMSF) from Boehringer–Mannheim, sodium fluoride (NaF) from Wako,

and sodium orthovanadate from Sigma. PMSF and pepstatin were dissolved in dimethyl sulfoxide (DMSO) to give 1 M and 1 mg mL−1 stock solutions, respectively, and the stock solutions were diluted 1000 times to produce solutions with final concentrations of 1 mM and 1 μg mL−1, respectively, and containing 0.1% DMSO. Leupeptin, aprotinin, and sodium orthovanadate were dissolved in pure water to give 1 mg mL−1, selleck inhibitor 1 mg mL−1, and 1 M stock solutions, respectively, and they were diluted 1000 times to produce final concentrations of 1 μg mL−1, 1 μg mL−1, and 1 mM solutions, respectively. NaF was dissolved in pure water to give a 200 mM stock solution for 200-times dilution to produce a final solution at 1 mM. All procedures were performed on ice or at 4 °C. The cells that had been stimulated to encyst for 1 h in a solution containing 1 mM Tris–HCl (pH 7.2) and 0.1 mM CaCl2 at a high cell density (50 000 cells mL−1) were collected by centrifugation (1500 g

for 2 min), rinsed in a macronuclei-isolation buffer [10 mM Tris–HCl (pH 7.2), 0.25 M sucrose, 3 mM CaCl2, and 10 mM MgCl2] (personal communication with Dr. Y. Kodama of Kochi University), and suspended in a buffer containing 0.2% Nonidet P-40 (NP-40). After being kept in this medium for 30–60 min, the cells were disrupted by pipetting; then, macronuclei were Megestrol Acetate collected by centrifugation (50 g for 5 min). To digest the sticky mucus-like materials that could cause co-adhesion of macronuclei, the pellets of the macronuclei were suspended in a macronuclei-isolation buffer (pH adjusted to 8.25 with NaOH) containing 10 mg mL−1 lysozyme (Sigma-Aldrich) for 1 h at 25 °C, followed by sedimentation of macronuclei by centrifugation (50 g for 5 min). For DAPI (4′, 6-diamidino-2-phenyl-indole

dihydrochloride) (Nacalai Tesque) staining, 2.5 μL of 0.02% DAPI (dissolved in pure water) was added to 0.5 mL of the suspension of macronuclei (final concentration of 0.0001%). After 5-min staining, the macronuclei were centrifuged (50 g for 5 min) and then suspended in a macronuclei-isolation buffer. The samples were observed under a fluorescence microscope (BX-50; Olympus) equipped with a WU filter set for DAPI staining. For immunofluorescence microscopy, cells were fixed with 2% paraformaldehyde in phosphate-buffered saline (PBS) for 1 h and rinsed with PBST (PBS containing 0.05% Tween-20). The cells were suspended in 1% NP-40 in PBS for 1 h, and subsequently transferred into PBS containing 1% polyoxyethylene (20) cetyl ether (Brij 58).