Somatostatin 14

As described previously (13 (link), 23 (link)), HEK 293T-Gα16gust44 cells were cultivated on poly-D-lysine coated 96-well-plates under standard conditions [Dulbecco’s Modified Eagle Medium (DMEM), 10% FCS, 1% penicillin/streptomycin, 1% glutamine; 37°C, 5% CO₂, saturated air humidity] and transiently transfected with expression constructs coding for the 25 TAS2Rs using lipofectamine 2000 (Thermo Fisher Scientific, Darmstadt, Germany). A transfection with empty expression vector was included for a negative control (mock). Duplicate wells were prepared for each compound-receptor combination. After transfection (∼24 h), cells were loaded with calcium-sensitive dye (Fluo4-AM, Thermo Fisher Scientific, Darmstadt, Germany) in the presence of probenecid (2.5 mM, Sigma-Aldrich, Steinheim, Germany) for 1 h, washed with C1 buffer (130 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 10 mM glucose, 10 mM HEPES; pH 7.4), stored in the dark for 30 min and finally washed once more. Cells were then placed in a fluorometric imaging plate reader (FLIPR^Tetra, Molecular Devices, San Jose, CA, USA). The bitter compounds, dissolved in the C1 buffer, were automatically administered to the cells and changes in fluorescence were monitored. For screening, we used 1 and 10 μM of lactucopicrin. Vitality of cells was evaluated by subsequent addition of somatostatin 14 (100 nM, Bachem, Bubendorf, Switzerland).

Cells were grown on coverslips for at least 24 h. For internalization studies, they were incubated with or without 1 µM somatostatin-14 (Bachem, Bubendorf, Switzerland) or octreotide (peptides & elephants, Hennigsdorf, Germany) in serum-free medium for 30 min at 37°C. Cells were washed with PBS, fixated in 1:1 acetone/methanol for 2 min, and incubated with anti-SSTR2 primary antibody (sc365502, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:50 in PBS overnight at 4°C. After washing with PBS, cells were incubated with a Cy3-conjugated goat anti-mouse secondary antibody (#115-165-146, Jackson ImmunoResearch, West Grove, PA, USA) diluted 1:1,000 in PBS for 1 h at room temperature. After washing with PBS, coverslips were briefly dipped into 96% ethanol, air-dried, and mounted on glass slides with Immu-Mount (Thermo Fisher Scientific, Waltham, MA, USA). Mounted cells were imaged using a confocal laser scanning microscope (LSM510, Carl Zeiss, Jena, Germany) using a helium–neon laser at 543 nm, LP560 emission filter, and 40× or 63× NeoFluar oil immersion objectives. RIN-1038-SSTR2-GFP cells were not immunostained after fixation, but otherwise treated the same. To visualize GFP fluorescence, an argon laser at 488 nm, LP505 emission filter was utilized on the same microscope.