D luciferin potassium salt

All experiments were approved by the Animal Ethics Committee of the University of Tokyo. The housing and handling conditions of the mice were consistent with the method above. Mouse renal orthotopic tumor models were conducted as previously described [22 (link), 29 (link)]. Briefly, BALB/c‐nu/nu male mice (5 weeks old) were purchased from Sankyo Labo Service Corporation (Tokyo, Japan). ccRCC cells (1.0 × 10⁵) expressing Luc2 and mCherry were inoculated into the subrenal capsule of mice. For in vivo bioluminescence imaging, D‐luciferin potassium salt (200 mg·kg⁻¹; Promega) was diluted in PBS and injected into mice intraperitoneally. For ex vivo bioluminescence imaging, the harvested kidneys and lungs were reacted with d‐luciferin potassium solution for 10 min, and images were captured using NightOWL LB981 (Berthold Technologies, Bad Wildbad, Germany). Quantitative analysis was conducted using the IndiGO software (Berthold Technologies). Everolimus was reconstituted in saline solution (Otsuka, Tokyo, Japan) containing 5% Tween20 and 30% propylene glycol (Sigma‐Aldrich) and administered to mice (2.5 mg·kg⁻¹) thrice weekly.

The rhythmic expression of PER2 was measured by using a real-time LUC assay in mouse embryonic fibroblasts (MEFs) derived from PER2::LUC knock-in mice24 (link). Bioluminescence was monitored once per minute over 10-min intervals with a dish-type luminometer (LumiCycle; Actimetrics, Wilmette, IL, USA). Cell lines were stimulated with 200 nM dexamethasone for 2 h to synchronize PER2 expression rhythms. The medium was replaced with DMEM containing 0.1 mM D-luciferin potassium salt (Promega, Madison, WI, USA), 10% FBS (Bio West, Kansas City, MO, USA) and the specified treatment before measurements were taken. For transient l-ornithine treatment, MEFs were treated with vehicle (water) or l-ornithine (1 mM) for 30 min at 36 h, 37 h, 43 h or 51 h after dexamethasone stimulation. After l-ornithine treatment, the l-ornithine-containing culture medium was replaced with fresh medium, and bioluminescence was measured over 4 days. The phase and amplitude of the second peak after treatment were recorded for analysis.

The tumour burden was quantified using whole-body in vivo bioluminescence imaging with a NightOWL LB981 system (Berthold Technologies). Mice were intraperitoneally injected with 2.5 mg of D-luciferin potassium salt (Promega) dissolved in 0.2 mL of PBS. Images were acquired beginning 10 min after D-luciferin injection. Regions of interest (ROIs) were drawn around the whole body. The photons emitted from the ROIs was quantified in units of photons per second using the IndiGO software (Berthold Technologies).

Tumor growth was monitored weekly using the IVIS Spectrum In Vivo Imaging System (PerkinElmer). Briefly, mice were injected subcutaneously with 75 mg/kg D-luciferin potassium salt (Promega, E1605) in sterile PBS and anesthetized with 2% isoflurane in medical air. Serial bioluminescence images were acquired using the automated exposure set-up. The peak bioluminescence signal intensity within selected regions of interest (ROI) was quantified using the Living Image Software (PerkinElmer), and expressed as photon flux (p/sec/cm²/sr). Representative planar bioluminescence images were displayed with indicated adjusted minimal and maximal thresholds.