Hek293 cells

HEK293 cells (American Type Culture Collections) stably expressing KORGFP, mycKOR, mycMOR, HA-D2DR, mycKOR and HA-D2DR, mycKOR and HA-PRDX6, or mycKOR (puro) and FLAG-Gαi3 were generated by transfecting HEK293 cells using Fugene HD (Promega) according to manufacturer instructions. HEK293 cells were maintained in Dulbecco’s modified medium/F12 (Fisher Scientific) with 10% fetal bovine serum (Sigma-Aldrich) and penicillin-streptomycin-L-glutamine (Fisher Scientific). HEK293 cells expressing mycKOR or mycMOR were grown in media supplemented with G418 (200 µg ml⁻¹) (Fisher Scientific); HEK293 cells expressing HA-D2DR were grown in media supplemented with hygromycin (50 µg ml⁻¹) (Fisher Scientific); HEK293 cells expressing mycKOR and HA-D2DR or HA-PRDX6 were grown in media supplemented with G418 (200 µg ml⁻¹) and hygromycin (50 µg ml⁻¹); HEK293 cells expressing mycKOR (puro) and FLAG-GNAI3 were grown in media supplemented with puromycin (0.5 µg ml⁻¹) (Sigma-Aldrich) and G418 (200 µg ml⁻¹). For SILAC cell culture, cells were grown for five doublings in 15 cm plates in custom DMEM media (Caisson Labs) containing isotope-labeled lysine and arginine (light media or L:R0K0 = lys0/arg0; medium media or M:R6K4 = lys4/arg6; heavy media or H:K10R8 = lys8/arg10) (Cambridge Isotope Labs).

The hAdam10 D + C domains (AA454–673) were amplified by PCR and cloned into the pDisplay vector (Thermofisher, Waltham, MA, USA) using BglII and SalI restriction enzyme sites (retaining the N terminal HA tag and Myc at the membrane). The construct was transfected into HEK293 cells (Fugene, Promega), and a stable line was generated through antibiotic (neomycin) selection.
For quantitative PCR, cDNA generated from RNA extracts (QIAGEN RNeasy) of snap-frozen tumor samples was analysed using an iTaq Universal SYBR Green kit (Bio-Rad Laboratories, Hercules, CA, USA) and a RotorGene 3000 cycler (Corbett Research). Primers specific for human ADAM10 were used to determine expression by comparative CT (ΔΔCT), relative to the averaged expression of three housekeeping genes (GAPDH, Beta 2 microglobulin, and Eukaryotic translation initiation factor 4E).

Gs22a cells are derived from HEK293 cells (ATCC CRL-1573) by stable transfection with the luciferase-based glosensor cAMP reporter plasmid pGlosensor-22F (Promega Corp.)^{28 (link)}. The cells were cultured in DMEM supplemented with fetal bovine serum (10%) in a humidified incubator containing 5% CO₂ and set at 37 °C, and seeded into 96- or six-well plates for transient DNA transfection and functional assays. The cells were transfected when the monolayers were 85–95% of confluency using Lipofectamine™ 2000 (ThermoFisher, Cat. No 11668019) and 100 ng of DNA per well for 96-well plates and 1000 ng of DNA per well for 6-well plates, except for DNA-titration studies, in which 150 ng of total DNA was used for 96-well plates (plate reader assays) and 2000 ng of total DNA was used for six-well plates (flow cytometry assays), with the total amounts of DNA being comprised of a quantity of receptor-expressing plasmid DNA ranging from 2 ng/well to 150 ng/well for 96-well plates, and from 60 ng/well to 2000 ng/well for six-well plates, and an appropriate quantity of pCDNA1 vector DNA such that the total amount of DNA was equal in all wells. Transfection mixtures were prepared in Opti-Mem™ (Gibco, Cat. No 31985070) and contained 3 μl of Lipofectamine™ 2000 per μg of DNA. Assays were performed 48 hours after transfection, and media was changed 2–4 h prior to assay.