Sciences). The signals were quantified using the ImageJ imaging software. Primary pituitary culture, electroporation and hormone assay Pituitary cell cultures were prepared from the anterior pituitary glands of 12- to 15-week-old male Wistar-Imamichi rats. The pituitary glands were finely minced and then digested by rapid agitation in DMEM containing 4 mg/mL collagenase (type 2; Worthington), 2 mM L-glutamine, 25 mM HEPES, and 400 mg/ mL DNase for 30 min at 37uC [68]. The pituitary cells were transfected using a microporator (Digital Bio Technology). In all, 1.56105 cells were electroporated with DNA (0.5 mg) or RNA (100 nM) at 1,500 V and a pulse width of 30 ms and seeded in 24- well plates (hormone assay) or 35-mm dishes (TIRF analysis). The cells were incubated in high-glucose DMEM containing 5% FBS and 15% horse serum at 37uC under 5% CO2. At 2 days after electroporation, the pituitary cells were preincubated in a buffer containing low amounts of K+ (140 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, 20 mM HEPES [pH 7.4] and 11 mM glucose) for 30 min, and then incubated in the same buffer for 24 h at 37uC. The levels of the hormones released into the culture medium were measured using radioimmunoassay kits for rat TSH (Shibayagi). The cells were harvested using celLytic buffer M (Sigma-Aldrich) containing protease inhibitors (Complete EDTA free; Roche Diagnostics). After removal of the cellular debris, the protein concentrations in the lysates were determined using the Bradford method. The pituitary cells were observed by TIRF confocal microscopy 1 day after electroporation. The plasma concentrations of mouse TSH were measured using radioimmunoassay kits (National Institute of Diabetes and Digestive and Kidney Diseases). TIRF microscopy For TIRF microscopy, a total internal reflection system (Nikon) was used with minor modifications. Light from an Ar laser (488 nm) was introduced into an inverted epifluorescence microscope through a single-mode fiber and a double-illumination lens. The cells were observed in the thermostat-controlled stage (37uC). PIP5K acetylation assay and in Vitro deacetylation assay HEK293T cells were transfected with Flag-PIP5Kc, Flag- PIP5Kc (K265/268R), Flag-PIP5Ka, or Flag-PIP5Kb and treated with 0 mM or 5 mM NAM (Sigma-Aldrich). After 24 h, the cells were placed in celLytic buffer M and immunoprecipitated. The Flag-PIP5Ks were eluted using the Flag peptide. The eluted Flag- PIP5Ks were combined with either recombinant SIRT1 or SIRT1 (H355A) in a reaction buffer (50 mM Tris-HCl [pH 8.8], 4 mM MgCl2 and 0.5 mM DTT), 100 mM of NAD+ was added, and the complexes were incubated for 3 h at 30uC as described [30]. The reaction mixture was used for measuring the PIP5Kc activity. Measurement of PIP5Kc activity The PIP5Kc activity was determined according to the procedure described in a previously reported [36]. Briefly, 0.1 pmol of PIP5Kc was incubated at 37uC for 25 min in 50 mL of 50 mM Tris–HCl, (pH 7.5), 1 mM EGTA, 10 mM MgCl2 and 0.004% (w/v) NP-40 containing 50 mM PI(4)P and 50 mM [c-32P]ATP (0.1 mCi/assay). After extraction using the Bligh and Dyer method, the lipids were separated by thin-layer chromatography (TLC) as previously reported [36]. The [32P]PI(4,5)P2 produced was analyzed with an FLA2000 Bio- imaging analyzer (Fuji Photo Film). SIRT1 Regulates TSH Release Lipid biochemistry The HEK293T cells or pituitary cells were spread on a 6-cm dish at concentrations of 56105 cells/well and the plates were incubated for 24 h. These cells were transfected or electroporated with SIRT1 stealth RNAi incubated with 4mCi of [32P]ortho- phosphate in phosphate-free DMEM. After 48 h of incubation, the lipids were extracted using chloroform:methanol:1N HCl (3:3:1, v/v). The lipids were dissolved in chloroform:methanol (2:1) before being spotted onto a TLC plate (silica gel 60; Merck). The plate had been sprayed with potassium oxalate, using a solvent system comprising 1.0% potassium oxalate in methanol:water (2:3, v/v), and pre-activated by heating at 110uC for 60 min before the spotting. The chromatogram was developed in chloroform:metha- nol:20% methylamine (60:36:10, v/v/v). The radioactivity was visualized using a Fuji FLA2000 bioimaging analyzer. Lipids were extracted from the mouse brain, corrected for tissue weight, and TLC was performed as described above. Each spot was visualized using 0.005% primuline, a fluorescent dye. The signals were quantified using ImageJ imaging software. Immunohistochemistry The isolated mouse and rat organs and the cultured cells were fixed with 4% paraformaldehyde and processed for immunostain- ing as described previously [69,70]. Human pituitary glands were obtained by concluding autopsies on the bodies of 20 individuals aged 20—103 years at the time of death. None of these individuals had a clinical history of abnormal endocrine dysfunction. Half of each pituitary gland was serially sectioned and the sections were scanned at a magnification of 40X. The total number of cells and the number of TSH-immunopositive cells in the entire area in each field-of-view were counted [71]. RT-PCR Total RNA was extracted using Sepasol reagent (Nakalai Tesque), precipitated by ethanol in the presence of ethachinmate (Nippon Gene), and reverse transcribed using ReverTraAce (TOYOBO). We used the following primers: 59-CGTCCCGTAGACAAAATGGT- 39 and 59-GAATTTGCCGTGAGTGGAGT-39 for mouse GAP- DH, 59-CGCATACGAGTGGAGAGAAA-39 and 59-ATGGC- GACAGGGAAGGAGAA-39 for mouse TSHb, 59-CAGCCT- GATGTTTGGTACCTCGGA-39. Signals were quantified using ImageJ software. Statistical analysis All the results are expressed as mean6SEM. The statistical significance of the differences between the groups was examined using Student’s t-test, Mann-Whitney U test, or repeated-measures analysis of variance (ANOVA), as appropriate, and the STAT- VIEW program. When a significant F ratio was obtained, we conducted post-hoc analysis by using Fisher’s protected least- significant difference (PLSD) test. P,0.05 was considered to be significant. Supporting Information Video S1 Real-time motion of EGFP-TSHb granules close to the plasma membrane of the pituitary cells was monitored at 5 s intervals by TIRF microscopy. Scale bar, 20 mm. Found at: doi:10.1371/journal.pone.0011755.s001 (0.06 MB AVI) Video S2 Real-time motion of EGFP-TSHb granules close to the plasma membrane of the SIRT1-overexpressing pituitary cells was monitored at 5 s intervals by TIRF microscopy. Scale bar, 20 mm. PLoS ONE | www.plosone.org 10 July 2010 | Volume 5 | Issue 7 | e11755