Victor 5

CHO cells stably expressing both the GPCR-omega chimera and ß-arrestin 2-alpha chimera were plated either in 6 well plates or in 96 well plates. After 24 h, ligands or HPLC fractions were added to the wells, and incubated for 14 h. After incubation, cells were washed once in HBSS and then incubated with CCF4/AM substrate for 2 h. ß-lactamase activity was measured either by FACS or with a plate reader (Victor V: Perkin Elmer).

Caspase activities were measured as described 39 (link)54 (link) in NP-40 lysates from cells triggered to undergo apoptosis or in cell-free cytosolic Jurkat extracts after cytochrome c/dATP-induced activation of the caspase cascade. Briefly, 10 µl cell extract was mixed in triplicate with 90 µl of 50 mM NaCl, 10 mM HEPES, pH 7.0, 40 mM β-glycerophosphate, 2 mM MgCl₂, 5 mM EGTA, 0.1 mg/ml BSA, 0.1% CHAPS and 10 µM Ac-DEVD-AMC (caspase 3/7 substrate) or 50 µM Ac-LEHD-AMC (caspase 9 substrate; both from Bachem, Weil am Rhein, Germany). Kinetics of substrate cleavage was recorded at 37°C using a Victor V fluorimeter (Perkin Elmer, Rodgau, Germany); the increase in substrate cleavage over time was used to calculate the caspase activity.

Cells harboring pDmpR-GESS or pmDmpR-GESS were cultivated in lysogeny broth (LB) medium (10 g tryptone, 5 g yeast extract, and 5 g NaCl per liter) and M9 minimal medium (12.8 g Na₂HPO₄·7H₂O, 3 g KH₂PO₄, 0.5 g NaCl, 1 g NH₄Cl, 2 mM MgSO₄, 0.1 mM CaCl₂, and 0.01% (w/v) thiamine per later) supplemented with 4 g/L acetate as a carbon source and 50 μg/mL ampicillin. For the two-step phenol reaction, the cells were grown in LB at 37°C until an OD₆₀₀ of 2.0 was reached, and then the culture media was changed to fresh M9 with 1 mM aromatic compounds and various concentrations of phenol by mild centrifugation (1,000 × g, 5 min) (Kwon et al., 2020 (link)). After 15 h of incubation at 37°C, the fluorescence intensities of the cells were measured using a FACSAriaIII (BD Biosciences, Franklin Lakes, NJ, USA) with a blue laser source (488 nm) and an FL1 (530/30 nm) photomultiplier tube. Data were acquired using BD CellQuest Pro (version 4.0.2, BD Biosciences) and analyzed using Flowjo software (Flowjo, Ashland, OR, USA).
To examine the antagonistic effect of pNP, cells harboring pDmpR-GESS were cultured in a two-step reaction in the presence of various concentrations of phenol and pNP. Fluorescence intensity and the optical density at 600 nm (OD₆₀₀) were analyzed with a multi-label reader (Victor V, PerkinElmer, Waltham, MA, USA).

CHO cells stably expressing the GPCR of interest were transiently transfected with NFAT-TA-Luc plasmid (Clontech). Cells were then left overnight before being plated in 96 well plates (20000 cells/well). After an additional 24 h, the synthetic ligands were added to the wells and incubated for 6 h. Following incubation, firefly luciferase assays were performed using the Steady-Glo Luciferase Assay System (Promega). Luminescence was detected with a plate reader (Victor V: Perkin Elmer), according to the manufacturer’s instructions. EC₅₀ values were calculated using Workout 2.0 Data Analysis Software (Perkin Elmer).

NA activity of the SH2 viruses was evaluated by the MU-NANA assay as previously described (47 (link)). Briefly, 50 μl of 20 μM 4-methylumbelliferyl-N-acetylneuraminic acid (MU-NANA; Sigma, St. Louis, MO) was added to the same volume of sample containing 32 HAU virus to incubate for 1 h at 37°C. The control (PBS [pH 7.4]) and a serially diluted standard (NA from Vibrio cholerae; Sigma, N7885) were incubated in parallel. Reactions were stopped with 0.1 M glycine (pH 10.7) in 25% ethanol, and the fluorescence was measured on an automated plate reader Victor V (Perkin-Elmer, Waltham, MA) at an excitation of 355 and an emission of 460 nm for 0.1 s per well. The viral NA activity was calculated using the standard curve of the Vibrio cholerae NA.

After pulse-chase labeling of HT-GPCRs, GPCR ligands were added to the wells and incubated for 1 h. Following incubation, cells were washed three times in PBS and fixed in 4% paraformaldehyde (PFA) for 30 min at RT. After washing, fixed cells were incubated with SA (Pierce) (5 μg/ml) for 20 min at RT. Cells were then incubated with 0.3% Triton X-100 for 10 min at RT to permeabilize the cell membrane. To eliminate internal peroxidase activities in the permeabilized cells, cells were treated with 0.3% H₂O₂/PBS for 30 min at RT. Cells were then incubated with SA-HRP (0.2 μg/ml) for 20 min at RT. After removal of unreacted SA-HRP by washing three times in PBS, cells were incubated with HRP substrates for 20 min at RT. The reaction was stopped by adding 2 N H₂SO₄ before measuring the optical density (OD) at 450 nm with a plate reader (Victor V, Perkin Elmer).

MDAMB-231 cells were obtained from American Type of Culture Collection. Changes in the cellular viability of compound 1-treated cells were monitored using the MTS activity assay (20). Briefly, MDAMB-231 breast carcinoma cells were seeded at day 0. After 24 h, complete medium was changed and compound 1 was added at different concentrations (starting with 50 µM containing 0.1 % dimethyl sulfoxide (DMSO) as vehicle). Doxorubicin at 8 µM was used as a positive control. After 48 h of incubation, incubation medium was removed and fresh medium with MTS (Promega)/phenazine methosulfate as electron coupling agent mixed solution was added. Lastly, optical absorbance was determined at 490 nm using a microplate reader (Perkin Elmer Victor V) according to the user manual. Additionally, any morphological changes associated with compound 1 and doxorubicin treated breast cancer cells were recorded under an inverted microscope after fixing the cells with trichloroacetic acid and cellular protein was stained with sulforhodamine B after 24 h (Kok et al. 2007 (link); Lam et al. 2015a (link), b (link)).