Beetle luciferin

Anti-BMAL1 (ab3350), anti-H3K27ac (ab4729), anti-H3K4me3 (ab8580), anti-H3K9me3 (ab8898) and anti-GAPDH (ab8245) antibodies were purchased from Abcam (Cambridge, UK). Anti-REV-ERBα (14506-1-AP), anti-DBP (12662-1-AP), anti-SLC1A5 (20350-1-AP), and anti-Histone H3 (17168-1-AP) antibodies were purchased from Proteintech (Wuhan, China). Anti-PPAR-γ (2443), anti-H3K9ac (9649), anti-H3K9me2 (4658), anti-H3K27me3 (9733) and anti-rabbit IgG (2729) were purchased from Cell Signaling Technology (Danvers, MA). Glutamine, methionine, C646 and MM-102 were purchased from Tsbiochem (Shanghai, China). [¹³C₅]-glutamine and [²H₃]-methionine were purchased from Zzbio (Shanghai, China). Dexamethasone was purchased from Sigma-Aldrich (St. Louis, MO). RNAiso Plus reagent, PrimeScript RT Master Mix and ChamQ Universal SYBR qPCR Master Mix were purchased from Vazyme (Nanjing, China). Beetle luciferin was purchased from Promega (Madison, WI). siRNAs for Slc1a5 and Ppar-γ, and negative control were purchase from IGE Biotechnology (Guangzhou, China). pcDNA 3.1, pcDNA 3.1-Ppar-γ, pcDNA 3.1-Slc1a5, pRL-TK, pGL4.0 and Slc1a5 luciferase reporters (-1400/+100 bp, -1000/+100 bp and a PPRE-mutated version) were obtained from Kaile Technologies (Guangzhou, China).

Beetle luciferin and potassium salt (Promega) were dissolved in PBS to a final concentration of 15 mg·mL⁻¹ and then filtered using a 0.22 μm filter. The luciferin solution was injected at 10 μL·g⁻¹ of mouse weight into each mouse intraperitoneally. After 10 min, bioluminescence was imaged using an IVIS Lumina II system (PerkinElmer) [18 (link)].

Images were acquired using IVIS SpectrumCT (Perkin Elmer). This multimodality imaging system allows the detection of tumors and metastases in X‐ray tomography coregistered with optical images of tagged tumor cells without image adjustment for anatomical correspondence. As light is only emitted by tumor cells without any background signal, bioluminescence is a highly specific and sensitive methodology for tumor detection and follow‐up over time 13. For optical detection, mice were injected intraperitoneally with 150 mg/kg of D‐luciferin (Beetle luciferin, Promega, Charbonnières, France) and then anesthetized with 3% isoflurane. For primary tumor detection, the lower section of the body (area of the lower legs) was imaged. For metastatic spread, especially lung metastases, primary tumor was covered to exclude its signal and chest was imaged. For primary tumors as for metastases, acquisition parameters were automatically computed by the SpectrumCT software to optimize bioluminescence signals (photons per second [p/s]) detection.

A kidney was collected from PER2::Luc mouse (12 weeks old, male), and sliced using a linear slicer (250 μm-thick) (Dosaka EM, Osaka, Japan). The slice was placed directly into the cell culture inserts (PICM ORG50, Millipore, MA, United States) in dishes with 1.2 ml of the culture medium containing phenol red-free DMEM (Nacalai Tesque, Kyoto, Japan), 10 mM HEPES (Nacalai Tesque, Kyoto, Japan), 1% Glutamax (Life Technologies, Carlsbad, CA, United States), 1 mM Sodium pyruvate (Nacalai Tesque, Kyoto, Japan), 2% B-27 supplement (Life Technologies, Carlsbad, CA, United States), 200 μM beetle luciferin (Promega, Madison, WI, USA), 100 units/ml penicillin, and 100 mg/ml streptomycin (Nacalai Tesque, Kyoto, Japan). The culture dish was set in the high-sensitivity charge-coupled device (CCD) camera-based microscopic imaging systems (Olympus, Tokyo, Japan; ATTO, Tokyo, Japan) and images were taken every hour. Further analysis was performed using AquaCosmos software (Hamamatsu Photonics, Hamamatsu, Japan).

For mouse experiments, female C57BL/6J mice were ordered from Janvier at 9–11 weeks of age. 1 million KPC or KPC-HAPLN1 cells expressing luciferase and RFP were injected intraperitoneally (i.p.) into mice. At day 8 and 10 of tumor growth, the belly of mice was shaved and Beetle luciferin (Promega, E1603) was injected i.p. 20 min after injection, luminescence was measured using an IVIS® Lumina LT In Vivo Imaging System. In total, tumors grew for 11 days. Mice were euthanized by rapid cervical dislocation. Peritoneal lavage was performed to remove cells suspended in the peritoneum by injection and subsequent removal of PBS into the peritoneal cavity after euthanization. Solid tumor masses formed in tumor cell injected mice, their size was always under the regulation limit (10 mm of diameter). For flow cytometry and cell sorting, solid tumors were digested using 2,5 mg/ml collagenase D (Sigma Aldrich, 11088866001), 0,5 mg/ml Liberase DL (Sigma Aldrich, 5466202001) and 0,2 mg/ml DNase (Sigma Aldrich, 10104159001) in 10% FCS DMEM for 45 min at 37 °C after mincing thoroughly. If peritoneal lavage contained erythrocytes, red blood cell lysis was performed with RBC Lysis Buffer (Life Technologies, 00-4333-57).

As shown in Fig. 1B, ATP measurements were performed at D0, D7 and Re-D7. At first, cell sheets gained by cultivation of OECs derived from luc tg rats were washed 3 times in HBSS, and incubated in HBSS for 1 hour at 4 °C. Then, beetle luciferin (Promega Corporation, USA) was added at a final concentration 0.19 mg/mL and photon intensity in the cell sheet was immediately measured before preservation based on the luciferase/luciferin reaction (to estimate the ATP levels) using a luminescence microplate reader (Centro LB960; Berthold Technologies GmbH & Co. KG. After each measurement, each cell sheet was washed 3 times in HBSS, immersed in the preservation medium, and left for 7 days at 4 °C.
The cell sheets were washed 3 times in HBSS after preservation, and the amount of ATP was measured (for comparison at D0 and D7). The medium was changed to KCM after ATP measurement at 7 days of preservation, and the sheets were re-cultivated for 7 days at 37 °C and 5% CO₂. After 7 days of re-cultivation, the amount of ATP was measured again. The ATP levels were calculated as the ratio (%) of ATP levels measured before preservation.