Slit2

The cells of each group were lysed in lysis buffer (#P0013B, Beyotime Biotechnology, China). Determine the quality of the harvested protein by using bicinchoninic acid (BCA) kit (#P0012, Beyotime Biotechnology, China). Then, 20 μg of total proteins was separated on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred on polyvinylidene difluoride (PVDF) membrane (#ISEQ00010, Millipore, USA) using the semi-dry transfer method. The membranes were blocked for 1 h in Tris-buffered saline containing 5% non-fat dried milk at room temperature (RT) and incubated overnight at 4°C with the relevant antibodies: SLIT2 (#47600), GFP (#55494), p-P38 MAPK (#4511), T-P38 MAPK (#8690), p-C-Fos (#5348), T-C-Fos (#2250), and β-actin (#4970) (1:1,000, all from Cell Signaling Technology, USA). Membranes were rinsed and incubated for 1 h with the corresponding peroxidase-conjugated secondary antibodies (#ab205718, #ab205719, Abcam, USA). Chemiluminescent detection was performed using the enhanced chemiluminescence (ECL) kit (#1251473, Thermo Fisher, USA). Bands were analyzed using ImageJ software (version 1.6 NIH) to verify the relative levels of the above markers. Results are representative of three independent experiments.

BRCA tissues (n = 20) or normal tissues (n = 4) of breast were obtained from the Affiliated Hospital of Guangdong Medical University. This study was approved by the Ethical Review Board of the Affiliated Hospital of Guangdong Medical University (#PJ2019-017KT). Informed written consent was obtained from each patient or their guardians. Immunohistochemistry (IHC) staining was performed on formalin-fixed paraffin-embedded (FFPE) tumor tissue sections. The tumor tissues were fixed in 10% formalin, embedded in paraffin, and serially sectioned into 4-µm-thick sections for histopathological study. Sections were deparaffinized in xylene, rehydrated in absolute and 90% ethanol serially, and washed with distilled water. After antigen retrieval in citrate buffer (0.01 M, pH 6.4), they were incubated in blocking solution (5% horse serum, 3% bovine serum albumin (BSA), 0.1% Tween-20 in PBS) for 1 h at RT, and then stained with antibodies against SLIT2 (Cell Signaling Technology, USA) at 4°C overnight in a humidified chamber. Sections were then washed several times with PBS and incubated 1 h at RT with the secondary antibody. All sections were counterstained with hematoxylin, dehydrated and mounted, and processed with peroxidase-conjugated avidin/biotin and 3′-3-diaminobenzidine (DAB) substrate (Leica Microsystem). IHC images were independently analyzed blindly by three pathologists.