In presence of saturating Ca2+ concentration (determined after the addition of 10 mL of digitonin solution (2 in water; Sigma), which caused lysis of the cells); Rmin: ratio in absence of free Ca2+, caused by addition of 50 mL of EGTA solution (600 mM in 1 M Tris buffer, pH 8.7) to lysed cells; SFB: correction factor; ratio of the fluorescence intensity (lex = 380 nm, lem = 510 nm) of the Ca2+ free and Ca2+ saturated dye.AutoradiographyAbout 4 million MCF-7 (L) cells (173rd in vitro passage, suspended in 0.1 mL of PBS) were subcutaneously injected into 12 female NMRI (nu/nu) mice bearing subcutaneous 17 stradiol depots [30] (implanted 14 days before). After 4 weeks of tumor growth, 6 animals, bearing tumors of comparable size (mean tumor area about 766 mm), were selected for control (3 mice) and tamoxifen treatment (3 mice). In case of the tamoxifen group, estrogen depots were explanted prior to tamoxifen administration. Tamoxifen citrate (12 mg/kg, dissolved inNPY Y1 Receptor Down-Regulation by AntiestrogensFigure 5. Proliferation of MCF-7 cells is unaffected by NPY. Effect of pNPY on the growth of MCF-7 (L) cells compared to the control. In all experiments the culture medium was supplemented with 17b-estradiol (1 nM). doi:10.1371/journal.pone.0051032.gPEG400/1.8 NaCl 1:1 at a concentration of 2.4 mg/mL) was injected subcutaneously on day 2, 6 and 10. The control group was treated with the vehicle. 14 days after removal of the estrogen depots, tumors were excised, immediately frozen in Tissue-Tek and stored at 278uC. Cryosections (12 mm) were obtained at 216uC with a 2800 Frigocut E freezing microtome (ReichertJung/Leica, Germany). Adjacent sections were mounted on three microscopic slides (Superfrost Plus, 7562561 mm) and kept in a chamber of 100 humidity for 1? min. Two slides were used to determine total and non-specific binding, and the third slide immersed in an 1531364 alcoholic formaldehyde fixative (37 (w/w) formaldehyde (40 mL), 95 (v/v) ethanol (360 mL) and calcium acetate (0.2 g)) for 20 s. For total binding the sections were covered with binding buffer (ca. 800 to 1000 mL) containing [3H]UR-MK114 (3 nM), and for unspecific binding with binding buffer, containing the radioligand (3 nM), pNPY (300 nM) and BIBP3226 (30 nM). The sections were incubated at room Ornipressin manufacturer temperature (22?5uC) for a period of 8 min. After incubation, the binding buffer was removed, the slides immersed three times into ice-cold buffer split to 3 vessels (each 10 s) and finally immersed into ice-cold demineralised water (3 s). The slides were put uprightly on a paper towel for 1 min and then dried in horizontal position in a desiccator over P4O10. The slides were set in close contact with a tritium sensitive screen (PerkinElmer, 1926125 mm) using an X-ray film cassette and stored in a dark room for 15 d. The autoradiographic image was generated from the tritium screen using an imager (Cyclone Storage Phosphor System, (-)-Calyculin A chemical information Packard). The fixed sections were stained according to Masson-Goldner (Jerusalem’s modification) using Weigert’s iron-haematein (45 s), rinsing (H2Odemin), running tap water (10 min), differentiation with 200 mL of H2Odemin +20 mL of 2 M aq. hydrochloric acid (15 s), running tap water (10 min), rinsing (H2Odemin), 0.5 aq. phosphotungstic acid (15 s), running H2Odemin (10 min), acid fuchsine-Ponceau (30 s), 1 aq. acetic acid (36immersion), phosphoric acid-Orange G (5 s), 1 aq. acetic acid (36immersion), 0.2 light green (3.5 min), 1 aq. acetic a.In presence of saturating Ca2+ concentration (determined after the addition of 10 mL of digitonin solution (2 in water; Sigma), which caused lysis of the cells); Rmin: ratio in absence of free Ca2+, caused by addition of 50 mL of EGTA solution (600 mM in 1 M Tris buffer, pH 8.7) to lysed cells; SFB: correction factor; ratio of the fluorescence intensity (lex = 380 nm, lem = 510 nm) of the Ca2+ free and Ca2+ saturated dye.AutoradiographyAbout 4 million MCF-7 (L) cells (173rd in vitro passage, suspended in 0.1 mL of PBS) were subcutaneously injected into 12 female NMRI (nu/nu) mice bearing subcutaneous 17 stradiol depots [30] (implanted 14 days before). After 4 weeks of tumor growth, 6 animals, bearing tumors of comparable size (mean tumor area about 766 mm), were selected for control (3 mice) and tamoxifen treatment (3 mice). In case of the tamoxifen group, estrogen depots were explanted prior to tamoxifen administration. Tamoxifen citrate (12 mg/kg, dissolved inNPY Y1 Receptor Down-Regulation by AntiestrogensFigure 5. Proliferation of MCF-7 cells is unaffected by NPY. Effect of pNPY on the growth of MCF-7 (L) cells compared to the control. In all experiments the culture medium was supplemented with 17b-estradiol (1 nM). doi:10.1371/journal.pone.0051032.gPEG400/1.8 NaCl 1:1 at a concentration of 2.4 mg/mL) was injected subcutaneously on day 2, 6 and 10. The control group was treated with the vehicle. 14 days after removal of the estrogen depots, tumors were excised, immediately frozen in Tissue-Tek and stored at 278uC. Cryosections (12 mm) were obtained at 216uC with a 2800 Frigocut E freezing microtome (ReichertJung/Leica, Germany). Adjacent sections were mounted on three microscopic slides (Superfrost Plus, 7562561 mm) and kept in a chamber of 100 humidity for 1? min. Two slides were used to determine total and non-specific binding, and the third slide immersed in an 1531364 alcoholic formaldehyde fixative (37 (w/w) formaldehyde (40 mL), 95 (v/v) ethanol (360 mL) and calcium acetate (0.2 g)) for 20 s. For total binding the sections were covered with binding buffer (ca. 800 to 1000 mL) containing [3H]UR-MK114 (3 nM), and for unspecific binding with binding buffer, containing the radioligand (3 nM), pNPY (300 nM) and BIBP3226 (30 nM). The sections were incubated at room temperature (22?5uC) for a period of 8 min. After incubation, the binding buffer was removed, the slides immersed three times into ice-cold buffer split to 3 vessels (each 10 s) and finally immersed into ice-cold demineralised water (3 s). The slides were put uprightly on a paper towel for 1 min and then dried in horizontal position in a desiccator over P4O10. The slides were set in close contact with a tritium sensitive screen (PerkinElmer, 1926125 mm) using an X-ray film cassette and stored in a dark room for 15 d. The autoradiographic image was generated from the tritium screen using an imager (Cyclone Storage Phosphor System, Packard). The fixed sections were stained according to Masson-Goldner (Jerusalem’s modification) using Weigert’s iron-haematein (45 s), rinsing (H2Odemin), running tap water (10 min), differentiation with 200 mL of H2Odemin +20 mL of 2 M aq. hydrochloric acid (15 s), running tap water (10 min), rinsing (H2Odemin), 0.5 aq. phosphotungstic acid (15 s), running H2Odemin (10 min), acid fuchsine-Ponceau (30 s), 1 aq. acetic acid (36immersion), phosphoric acid-Orange G (5 s), 1 aq. acetic acid (36immersion), 0.2 light green (3.5 min), 1 aq. acetic a.
