D luciferin substrate

Female athymic BALB/c nu/nu mice about 4-6 weeks old were used for the experiment. The use of animals was approved by the ethical committee of Xi'an Jiaotong University. MIA PaCa-2 cells with RASAL2 overexpression (Lv-RASAL2) and control cells (Lv-NC) were prepared for further use. For subcutaneous xenotransplanted tumor model, six mice per group were randomly distributed into two groups (Lv-RASAL2 and Lv-NC), 100 µl serum-free DMEM medium containing Matrigel (1:1, v/v) and 1×10⁶ cells were injected subcutaneously into flanks of the mice. About 30 days after tumor cell injection, the mice were sacrificed and xenograft tumors were harvested, weighed and processed for immunohistochemistry staining. For tail-vein injection model of cancer metastasis, ten mice per group were randomly distributed into two groups (Lv-RASAL2 and Lv-NC), 2×10⁶ cells were suspended in 200 µl serum-free DMEM and injected via the tail vein. About 6 weeks after tumor cell injection, D-luciferin substrate (Biosynth, Naperville, IL, USA) in PBS with 450 mg/kg was injected into peritoneal cavity; then 15-20 minutes later, mice were anesthetized and bioluminescence imaging (BLI) was performed to detect the distant metastases in the lung and other organs.

The tail-vein injection metastasis model was generated as described in previous studies. Wild-type (WT) (C57/BL6J) mice (8 weeks) were used according to protocols approved by the Ethics Committee of Xi’an Jiaotong University. Piezo1-silenced B16 melanoma cells with luciferase markers (1.5 × 10⁵ cells, 100 μl) were suspended in serum-PBS and introduced into the circulation of mice by tail-vein injection. After 6 weeks, 150 mg/kg D-luciferin substrate (Biosynth, Naperville, IL, USA) in PBS was injected into the abdominal cavity. Fifteen to 20 min later, bioluminescence imaging (BLI) was performed to detect distant metastases in the lung and other organs after the mice were anesthetized. The mice were then sacrificed, the lungs were lavaged with PBS to clear them, and the number of melanoma metastases was counted under a microscope (Invitrogen, USA).