Robo1

4% formaldehyde was used for section fixation, and 1 mM EDTA (pH 8.0) was applied for antigen repair. The sections were incubated with goat serum for 2 h at room temperature, followed by overnight incubation with primary antibody (Abcam, ROBO-1, ab256791) at 4 °C. The sections were then incubated with the secondary antibody and then reacted with diaminobenzidine and counterstained with Mayer hematoxylin.

Human osteoblast hFOB1.19 cells and human osteosarcoma Saos-2 and MNNG-HOS cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Human osteosarcoma U-2OS cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Each cell line passed the test of DNA profiling (STR). Mycoplasma contamination testing was performed using Mycoplasma Genus PCR. All cell lines were cultured according to ATCC instructions. Standard culture conditions were used for the human osteosarcoma cells (37 °C in a 5% CO₂ atmosphere), while the hFOB1.19 cells were incubated at 34.5 °C in a 5% CO₂ atmosphere. Cells were passaged 7–10 times. The antibodies used were ROBO1 (ab7279; Abcam, Cambridge, UK), SLIT2 (ab134166; Abcam), SRC (2109; Cell Signaling Technology, Danvers, MA, USA), p-Src (12432; Cell Signaling Technology), extracellular signal-regulated kinase (ERK, 4695; Cell Signaling Technology), p-ERK (4370; Cell Signaling Technology), c-Myc (ab32072; Abcam), PFKFB2 (17838; Proteintech, Wuhan, China), β-actin (M1210-2; Hua’an Biology, Chuzhou, China), BrdU (66241-1-1g, Proteintech, Wuhan, China), horseradish peroxidase-conjugated anti-rabbit IgG (170-6515; Bio-Rad Laboratories, Hercules, CA, USA), and horseradish peroxidase-conjugated anti-mouse IgG (170-6516, Bio-Rad Laboratories).