Coelenterazine h

A substrate for GLuc, coelenterazine h, was purchased from Wako Pure Chemicals. HEK293T cells (5 × 10⁵ cells per well) were cultured in 6-well dishes for 24 h, and transiently transfected with plasmids encoding CCR7-NGLuc and CCR7-CGLuc using PEI-MAX. After 24 h, the cells were cultured in fresh media for a further 24 h and subjected to the luminescence measurement. Five minutes after adding coelenterazine h (20 μM) to phenol red-free culture medium, luminescence signals were integrated over 2 sec using a LB9507 luminometer (Berthold Technologies, Bad Wildbad, Germany) or over 10 sec using an IVIS system (Perkin Elmer, MA, USA).

A 3 mL volume of reporter T87 cell culture of 150 μL PCV suspended in NT1 medium containing 2% v/v FP003 solution (FCeM Advance Preparation Kit500; Nissan Chemical Industries, Tokyo, Japan) was transferred to a quartz cuvette. Three hundred μL of Nano-Glo luciferase assay reagent (Promega) or 60 μL of 5 mM coelenterazine-h (Fujifilm Wako) were added to the cell culture and mixed. Luminescence spectra were immediately measured using an FP-8300 spectrofluorometer (Jasco, Tokyo, Japan) at 25°C with the excitation light source turned off (emission bandwidth, 5 nm; scan speed, 1000 nm/min). Spectral data were scanned 20 times and integrated to reduce the noise. The spectra thus obtained were normalized to the peak height for each reporter cell type in the range of 400 to 700 nm. The BRET ratio is defined as the ratio of acceptor (GeNL: 520 nm) and donor (NLuc: 450 nm) luminescence intensities.

A luminescent calcium assay using aequorin as an indicator was performed according to the previous reports (84 , 85 (link)). 293T cells were seeded into white-walled 96-well plates (MS-8096W, Sumitomo Bakelite) at 20,000 cells per well in Dulbecco's modified Eagle's medium/F-12 (FUJIFILM Wako) containing 10% FBS. After a day, cells were transfected with the expression plasmids of the opsin and the mitochondrial targeted aequorin N26D using polyethylenimine (PEI MAX, Polyscience). The ratio of polyethylenimine:plasmid was 4:1 in weight. The ratio of plasmids of opsin:aequorin (Figs. 2 and 3) and that of opsin:Gα:aequorin (Figs. 4 and 5) were 1:1 and 1:1:2 in weight, respectively. Six to eight hours after transfection, the medium was replaced with a fresh medium containing 2 μM 11CR or 5 μM ATR. The next day, the medium was replaced with an L-15 medium without phenol red (Invitrogen) containing 10% FBS and 10 μM coelenterazine h (FUJIFILM Wako) under dim red light. After 2 h of incubation in the dark, luminescence was measured using a microplate luminometer (Veritas, Turner Biosystems). The cells were stimulated with a handheld UV flashlight (0.11 ± 0.0036 mW mm⁻²; peak irradiance at 373 nm, Fig. S12) for 5 s.

Bioluminescence imaging with an IVIS was conducted as described previously.⁵⁷ (link) In brief, eight mice were infected with 1 × 10¹⁰ CFU STEC E32511 or O111 (four mice for each strain). After 1 or 2 days, mice were intravenously injected with nano-lantern-labeled Muse or non-Muse cells (1 × 10⁵/mouse).²⁵ (link) Mice were then intravenously injected with 100 μL substrate (80 μg/mL coelenterazine-h; Wako, Tokyo, Japan), anesthetized with isoflurane by inhalation, and placed in an acrylic plastic box. Bioluminescent images were acquired using an IVIS (Spectrum; PerkinElmer, Waltham, MA, USA) using the following settings: exposure time, 1 min; medium binning, F/Stop = 1. Data acquisition and analysis were performed using Living Image 4.4 software (PerkinElmer). Quantification was performed using a region of interest (ROI) defined manually (abdominal center or injection site), and the results are expressed as photons per second (P/s).

HEK293 cells were transfected with β₁AR-Rluc, βArr2-YFP or Gα_s-YFP plasmid Readings along with pCI-neo, TRPC6(WT), TRPC3(WT) or TRPC6(DN) vectors. At 48 h after transfection, cells were detached with PBS/EDTA (2 mM) and centrifuged for 5 min (1000 × g) at room temperature. After centrifugation, cell pellets were resuspended in BRET buffer (1.8 mM CaCl₂, 1 mM MgCl₂, 0.1% glucose in PBS) and moved to 96-well white microplates. Cells were stimulated with ISO (10 μM) for 30 min and incubated with coelenterazine h (5 μM, Wako) for 10 min. The BRET signal was determined as the ratio of the light emitted by YFP and the light emitted by Luc using a fluorescent microplate reader (SpectraMax® i3, Molecular Devices). The values were corrected by subtracting the background BRET signals.

Diacetyl Coelenterazine-h was synthesized as previously described^{14 (link)}. Coelenterazine-h and PD-0325901 were obtained from Wako (Osaka, Japan). Gefitinib and AZD6244 were purchased from Symansis (Shanghai, China). Anisomycin, dbcAMP and epidermal growth factor were purchased from Sigma-Aldrich (St. Louis, MO). 293fectin Transfection Reagent was obtained from ThermoFisher Scientific and used for plasmid lipofection into HeLa cells. The Nano-Glo luciferase assay system was purchased from Promega (Madison, WI) and the assay substrate included in the kit was used as a stock solution of furimazine. The luciferins used in each luminescent assay are listed in Table S3.