G1014

For migration detection, after transfection for 24 h, cells (3 × 10⁵/mL) were cultured in medium (no serum). The final concentration of fetal bovine serum was 1% and the volume was 150 μL when cells were added to the upper chamber. We added 10% FBS medium in the lower chamber to make the volume 500 μL. After 48 h of incubation in the cell incubator, the upper chamber fluid was absorbed and placed in a 24-well plate containing 500 μL of 4% paraformaldehyde (G1101, Servicebio, China). The cells were fixed at room temperature for 20 min. After imbibing the fixative solution, the cells were placed in a 24-well plate containing 500 μL 0.1% crystal violet solution (G1014, Servicebio) for 20 min, and then the inner surface cells of the bottom membrane of the upper ventricle were wiped off. The results were observed and captured under the microscope. For invasion measurement [19 (link)], the apical chamber was coated with Matrigel matrix and then cells were added. The remaining steps were the same as those in migration detection.

NSCLC cells were pretreated with indicated CM for 48 hours. Appropriate numbers of cells were seeded into 6-well plates depending on the different radiation doses and exposed to 0, 2, 4, 6, and 8 Gy radiation. The plating efficiencies (PE) in different groups are shown in Supplemental Table 2. Cells were further allowed to grow for 10–14 days to form clusters, followed by 4% paraformaldehyde fixation and crystal violet (G1014, Servicebio) staining. Colonies comprising more than 50 cells were counted. The calculation formulae for PE and survival fraction (SF) are as follows: PE = number of colonies counted/number of colonies seeded. SF = (number of colonies counted/number of colonies seeded)_test/(number of colonies counted/number of colonies seeded)_control, where “test” denotes the test condition (some radiation dose) and “control” denotes identical cells without radiation.

Cells were seeded in six‐well plates at a concentration of 500 cells per well and cultured for 2 weeks in the presence of various concentrations of orexin‐A (0.1 μM). In the rescue experiments, Fer‐1 (5 μM) were added in cells 1 h before orexin‐A treatment. Colonies were fixed with 4% paraformaldehyde (Biosharp, Anhui, China) and stained with 0.1% crystal violet (G1014, Servicebio, Wuhan). Then, effective colonies were screened and counted.

In the clone formation assay, 400 cells were initially seeded per well in a 6-well plate. The experimental group received 1 µg/ml of PTEN-L, while phosphate-buffered saline (PBS) served as the control. After ten days, the culture medium was discarded, and cells were washed with PBS. Subsequently, cells were fixed with 4% paraformaldehyde (PFA, Solarbio, Beijing, China) for 15 min at room temperature, followed by staining with 0.1% crystal violet (G1014, Servicebio, Wuhan, China) for 10 min. Excess dye was removed by washing with 1×PBS. Cell colony images were captured using the ChemiDoc MP imaging system from Bio-Rad, Hercules, CA, USA, and colony quantification was performed using Image J software.

Approximately 10³ cells were seeded and cultured in a 6-well plate for 2 weeks. The cells were fixed with methanol, stained with 0.05% crystal violet (Servicebio, G1014, Wuhan, China), and colonies were observed.