Coelenterazine

Embryos were microinjected as described above with 200 pg of in vitro-transcribed zfiGLuc or GLuc RNA. After 48 h, larvae were infected with WT ST (MOI of 10) and whole-larval extracts were obtained at 24 hpi as described previously65 (link). Larval extracts were then combined 1:1 with distilled water containing 4.4 μM coelenterazine (Sigma-Aldrich) to achieve a final concentration of 2.2 μM. The luciferase signal was then measured on a Luminometer Optocomp II (MGM Instruments).

Twenty-four hours after transfection, the CHO cells were washed in PBS and afterwards resuspended in PBS + 1% glucose. Eighty µl of 4 × 10⁶ cells/ml were seeded in a 96-well plate (~100,000 cells/well). Five µM coelenterazine (NanoLight Technology, Pinetop, AZ, United States) was added to the cells. After 10 min, 5 µl of varying ligand concentrations was added to each well. After 15 min coelenterazine and 1 µM forskolin (Sigma-Aldrich) were added. The plates were kept in the dark all the time. The emission signals from Renilla luciferase (RLuc) and yellow fluorescent protein (YFP) were measured with a 2104 EnVision Multilabel plate reader (PerkinElmer, Waltham, MA, United States). The BRET signal is the ratio of the detected YFP (acceptor emission) at 525 nm divided by the RLuc at 475 nm (donor emission) [30 (link)].

The day before transfection, cells were trypsinized and transferred into wells of polystyrene 96-well culturing plates (Costar). Ten µL of a water solution containing 30 ng of the mutated Gal4-FusionRed-p65 construct, 0.1 µg of the pG5-Luc reporter (Promega), encoding the secreted Gaussia luciferase, and 0.2 µg pGl3-Basic vector was mixed with 1.5 µL of 1 mg/mL solution of PEI, pH = 7.0. The mixture was incubated for 10 min at room temperature for the efficient formation of DNA-PEI complexes and added directly to cells in plate wells containing 100 µL of the cultured medium. After transfection, cells were illuminated for 24 h with either blue pulsed light (array of 448 nm light-emitting diodes (LEDs), 30 s ON, 5 min OFF) or yellow-orange pulsed light (567 nm LED array, 30 s ON, 5 min OFF) at a light power density of 100 mW/cm².
For measurements of Gaussia luciferase activity, 1 µL of cell culture medium was mixed with 100 µL of 2 µM coelenterazine (Sigma, St. Louis, MO, USA) in wells of 96-well reflective bottom plates for bioluminescent assays (Stellar Scientific, Owings Mills, MD, USA). The quantification of bioluminescence was performed using a Victor X-5 plate reader (Perkin Elmer, Waltham, MA, USA). The contrast of 448 nm/567 nm reporter expression was calculated as luciferase activity in samples under illumination with 448 nm light divided by that at 567 nm light.

PHKs and PMKs were transfected with reporter constructs using a TransIT-X2 Dynamic Delivery System (Mirus Bio) according to the manufacturer’s instructions. Cotransfection of the Renilla luciferase expression vector pRL-TK (Promega) was used as an internal control for all reporter assays. Cell extracts were generated 24 hours after transfection using Reporter Lysis Buffer (Promega) and extracts were assayed for firefly luciferase and Renilla luciferase activity using the Luciferase Assay system (Promega) and coelenterazine (0.1 μg/mL, Sigma-Aldrich), respectively. Luminescence was measured with the Cytation3 Imaging Reader (BioTek).

Ethidium bromide (EtBr), ethylene glycol-bis (β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), ethylenediaminetetra acetic acid (EDTA), isopropyl-β-D-thiogalactoside (IPTG), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) coelenterazine, carbenicillin, CaCl₂, Carbonyl cyanide m-chloro phenylhydrazone (CCCP), Calmidazolium (CDZ), Verapamil, coelenterazine, Trifluoperazine (TPZ), Chlorpromazine (CPZ), Phosphate Buffer Solution (PBS), Triton X-100, Tris-Acetate EDTA buffer (TAE), and aequorin oligonucleotides were all purchased from Sigma-Aldrich (St. Louis, MO, United States). Taq polymerase master mix for PCR (Promega, Madison, WI, United States), agarose molecular grade and 100 bp molecular ruler were obtained from Bio-Rad, Hercules, CA, United States). Kanamycin, Gentamicin, Streptomycin, Vancomycin, Ciprofloxacin, and Erythromycin (St. Louis, MO, United States). BamHI and HindIII (Rowley, MA, United States), T4 ligase (Rowley, MA, United States).

Stable cell lines expressing the mussel receptors were transiently co-transfected with the mitochondrial targeted apo-aequorin protein cloned in pcDNA3.1. Two days after transfection cells were washed, counted and resuspended in 1 ml of DMEM /F12 Ham (without phenol red)/0.1% BSA supplemented with 2 µM coelenterazine (SIGMA-ALDRICH). Cell suspensions were incubated for 3 hours at RT in the dark with gentle agitation. To obtain a final concentration of 5 ×10⁵ cells/ml they were subsequently diluted in DMEM / F12 Ham / 0.1% BSA and incubated for an extra hour at RT in the dark with gentle agitation. The injector system of the Biotek Synergy 4 microplate reader (USA) was used to inject 50 µl of the cell suspensions into wells containing 50 μl of the diluted peptide in DMEM / F12 Ham / 0.1% BSA. Bioluminescence assays were measured using white, 96 well plates with a flat bottom (INVITROCELL, Portugal) and luminescence was recorded every 2 s for 30 s. Peptide induced Ca²⁺ responses were normalized to the total Ca²⁺ response after addition of Triton X-100 (0.1%). Peptide potency was determined as the total area under the activation curve. Data was calculated as a % of the highest response (100% activation). Assays were performed in duplicate and EC₅₀ calculated from dose–response curves obtained in three independent assays (GRAPHPAD PRISM, 7.0a).

Intracellular calcium levels were measured either with using calcium indicator Fluo-4 AM (Molecular Probes) according to the manufacturer’s instructions or with the calcium-activated photoprotein, aequorin, as described previously (15 (link)). In brief, for aequorin-mediated fluorescence measurements COS-7, HULEC5α or Cav-1 KO HULEC5α cells were transduced with adenoviruses encoding aequorin, RFP or Cav-1 at 15 MOI, and 24 h later the aequorin was reconstituted by treating cells with 5 μM coelenterazine (Sigma, St. Louis, MO) for 1 hour in serum-free and phenol free DMEM containing 0.1 mM EDTA. Then, the cells were exposed to different concentrations of PLY in Hank’s Balanced Soult Solution (HBSS) for 10 minutes, and luminescence was recorded using a PolarSTAR luminometer (BMG Labtech, Cary, NC). After the baseline luminescence values were recorded, calcium chloride was injected at a 1.8 mM final concentration to enable the measurement of calcium induced changes in luminescence which reflect calcium influx.