D luciferin substrate

Control or PRMT5‐overexpressing HT29 cells (5 × 10⁵ cells in 100 μL of PBS) were injected into the tail veins of 5‐ to 6‐week‐old BALB/c nude mice. Six weeks after injection, mice were anaesthetized with isoflurane (Sigma) and intraperitoneally injected with D‐Luciferin substrate (PerkinElmer, Waltham, MA, USA) prior to analysis of bioluminescence signals using an in vivo imaging system (IVIS) Spectrum System (Caliper Life Sciences, Waltham, MA, USA). After mice were sacrificed, lungs and brains were surgically excised and processed for staining with haematoxylin and eosin (H&E). All mouse studies and procedures were conducted in accordance with the guidelines established by the Animal Care and Use Committee of Zhejiang University.

To further explore the therapeutic efficacy of our therapeutic strategy, tumors were also assessed using an in vivo bioluminescence imaging system (Bruker Xtreme scanner). Mice were monitored for tumor growth by bioluminescent in vivo imaging every 6 days (Day 0, 6, and 12); specifically, 8 minutes after intraperitoneal injection of 150 mg/kg D-luciferin substrate (PerkinElmer, Catalog#122799), mice from each treatment group (n = 3) were imaged.

All in vivo imaging experiments were performed using Xenogen IVIS Spectrum imaging system (Caliper Life Sciences). Prior to imaging, mice were injected intraperitoneally with 100 µl of phosphate-buffered saline containing d-Luciferin substrate (PerkinElmer). For biodistribution study, 100 µl of ZnS-QDH solution was injected into the tail vein of tumor bearing mice and the fluorescent imaging data were collected at 24 and 48 h after injection. After 48 h, animals were euthanized and selected major organs (liver, kidney, spleen, lung, and heart) were extracted for ex vivo imaging. Tumor growth was measured both by caliper measurement and bioluminescence imaging.

Four-week-old female nude mice were purchased from Vital River Company (Beijing, China) and maintained in Specific Pathogen Free (SPF) animal facility (68–71.6 °F temperature and 50–60% humidity). 8 × 10⁵ MGC803 cells constitutively expressing luciferase cells in 0.2 ml PBS were injected into the tail vein of mice. Ten days later, pentobarbital-anesthetized mice were injected intraperitoneally with the d-luciferin substrate (PerkinElmer, USA; 15 mg/ml in PBS) and imaged under a Bruker In-Vivo Xtreme system to observe the metastasis. For all analyses, an additional ROI was employed to normalize for background luminescence on each image. After the metastasis model was established in most mice, they were randomly divided into three groups (6 mice per group). 14-5-18 was dissolved in a mixed solvent of DMSO (2%) and Tween 20 (5%) in water, and administrated by gavage at 0, 10, and 30 mg/kg for each group, once a day. After 18 days of administration, the mice were imaged with a Bruker In-Vivo Xtreme system. Mice were sacrificed, and their lungs were removed and stained by Hematoxylin and Eosin (H & E) staining.

BALB/c nude mice (4 weeks old) were maintained in a specific pathogen-free (SPF) environment. Animal welfare and the experimental procedures were in compliance with the National Institutes of Health Guidelines for the care and use of laboratory animals, and all procedures involving the mouse xenograft model were approved by the Ethics Review Committee of Nanjing University of Chinese Medicine. The luciferase-expressing A549 cell line with lentivirus was established to generate the orthotopic xenograft lung cancer model. A549 cells (2 × 10⁷ in 0.2 ml medium of a 1:1 mixture of RPMI 1640 and Matrigel 354248) were then injected into the right lung parenchyma of mice. Two weeks later, the mice were randomly divided into an OP-B-treatment group (2.5 mg/kg OP-B, n = 6) and a control group (treated with saline, n = 6). Treatment was administered to all mice via intraperitoneal (i.p.) injection (daily). The volume and the metastasis of tumors were determined by in vivo bioluminescence measurements (IVIS Spectrum, PerkinElmer, Waltham, MA, USA) after an i.p. injection of 200 μl d-Luciferin substrate (15 mg/ml in Dulbecco’s PBS; PerkinElmer, Waltham, MA, USA). On day 45 of i.p. injection, all tumors were collected and the following tests were carried out.