Nightowl 2 lb 983 system

All experiments were performed according to guidelines of Institutional Animal Committee of Kumamoto University. For intracardiac injections, subconfluent tumor cells were harvested, washed in PBS, and resuspended at a concentration of 1 × 10⁶ cells/ml. Non obese diabetic/severe combined immunodeficient (NOD/SCID) Janus kinase 3 knockout (NOJ) mice26 (link) were anesthetized by isoflurane and placed in the supine position. With a 29-gauge needle, 1 × 10⁵ cells were injected into the left ventricle of anesthetized mice after visualization of arterial blood flow into the syringe. Metastasis was monitored by bioluminescence imaging for 5 weeks after injection. D-Luciferin (100 μg/g) was injected into back skin of anesthetized mice before imaging, and images were then acquired using a NightOWL II LB 983 System (Berthold Technologies, Bad Wildbad, Germany). Luminescence was calculated using IndiGO2 software. For the orthotopic implantation model, subconfluent tumor cells were harvested, washed in PBS, and resuspended at 1 × 10⁷ cells/ml. Female 8–12 week-old NOJ mice were anesthetized by isoflurane, and for the breast cancer model, cells (1 × 10⁶) in 100 μl PBS were injected into their mammary fat pads.

The protocols of the Institutional Animal Care and Use Committee (IACUC) of the Affiliated Hospital of Qingdao University were used to guide the design of all animal studies, which received approval from the Affiliated Hospital of Qingdao University IACUC committee (AHQU‐MAL20190356). For these experiments, BALB/c athymic nu/nu mice (Vital River, 5 weeks old) received an injection in the left liver lobe of Huh7 cells (5 × 10⁶) stably expressing luciferase that had been transfected with siMock or siHULC constructs. Cells were injected in a 100 μl volume containing a 1:1 mixture of culture medium and growth factor‐reduced Matrigel. Orthotopic HCC xenograft tumor growth was measured every week after injection by injecting anesthetized mice with D‐Luciferin (100 μg/g), with a NightOWL II LB 983 System (Berthold, Bad Wildbad, Germany) then being used for murine imaging. The IndiGO2 software was used for luminescence calculations. At 4 weeks following tumor injection, nude mice were euthanized via carbon dioxide inhalation followed by cervical dislocation and the xenograft tumors were collected for histological and immunohistochemical staining.

Original vector containing Cas91.1 expression frame and gRNA editing region purchased from Addgene (#71814), gRNA designed online and cloned into vector Cas91.1, verified by sequencing. The final sequence of Nrf1 (XM_027775873.1) gRNA is 5′-GGATTAGACTCAAACACGTG-3′ according to its score calculated on https://www.genscript.com/gRNA-design-tool. Purified plasmid of the final version was transfected into cells by JetPEI reagent as described in above chapter.
The positive signal, e.g., EGFP was verified by fluorescence microscope. Also, the signal in living cells e.g., Luc activity can be examined in the 96-well culture plate by adding additional Luciferin substrate, and the signal was read by NightOWL II LB 983 system (Berthold Technologies).

BLI was performed at Vivoptic (Bordeaux University) using a NightOWL II-LB 983 system (Berthold Technologies, Bad-Wildbad, Germany). Mice received an intra-peritoneal injection of D-luciferin (Promega, 2.9 mg in 100 μL PBS) and were sedated 7 min later. Bioluminescence images (1 min, 4 × 4 binning) and photographs (100 ms) were taken 10 min after luciferin injection. The bioluminescence signal was analyzed using Indigo software. Prostates, removed from euthanized animals immediately after in vivo BLI, were placed in cold PBS on a glass slide and imaged (1 min). LNCaP-lucF cells in PBS in 12-well culture plates were imaged (1 min) by BLI immediately after adding D-luciferin (6.10⁻⁴M in 200 μL PBS).