Blocking solution

For histological assessment, the spleen, liver, and intestine were fixed in 4% paraformaldehyde and embedded in paraffin. Three micrometer sections were stained with hematoxylin and eosin (H&E). The entire images were captured by APERIO CS2 (Leica, Germany). For immunofluorescence staining, the spleens were frozen in OCT and cut into 8 μm sections. After being fixed by 4% paraformaldehyde at room temperature (RT) for 10 min, the sections were treated Blocking Solution (Beyotime, China) at RT for 1 h. Then, the samples were probed with the primary antibody Anti-F4/80 (1:150, Abcam, Cambridge, UK) at 4 °C overnight, then incubated with the secondary antibody DyLight 550 donkey anti-rat IgG (H + L) Cross Adsorbed (1:500, Life Technologies, Carlsbad, CA, USA) at RT for 1 h. Cell apoptosis was detected using TUNEL staining kit (KeyGEN BioTECH, Nanjing, China) according to the manufacturer’s instructions. The fluorescence was observed by confocal microscopy. The apoptotic cells of macrophages were quantitatively analyzed (apoptotic cells/F4/80⁺ area) by software ImageJ.

Proteins were isolated from transfected cells by RIPA lysis buffer (Beyotime, Nantong, China) containing 0.5% PMSF. The total proteins (50 μg per protein) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated for 1 h in blocking solution (Beyotime) at room temperature and then immunoblotted overnight at 4°C with the following primary antibodies: anti-MDMX (1:1,000, Proteintech Group, Rosemont, IL, USA), anti-P53 (1:1,000, Proteintech Group, Rosemont, IL, USA), anti-P21 (1:1,000, Proteintech Group, Rosemont, IL, USA) and anti-GAPDH (1:1,000, Beyotime). And enhanced chemiluminescence reagent kit (Millipore, Billerica, MA, USA) was utilized for exposure after the blot incubated with secondary antibody (Beyotime) for 1 h.

The paraffin-embedded lung sections were dewaxed and cleared with xylene, followed by hydration with gradient ethanol. Then, the sections were retrieved with sodium citrate solution (pH 8.0; Beyotime, Shanghai, China) for 20 min. The sections were blocked with blocking solution (7.5% goat serum and 0.5% Triton X-100 in PBS; Beyotime, Shanghai, China) for 1 h and incubated with primary rabbit (anti-CD31, Abcam, ab6994, 1:200) anti-Sema7a (Abcam, ab23578, 1:200), anti-RAGE (Abcam, ab21171, 1:200), and anti-Ki67 (Abcam, ab15580, 1:1000) antibodies overnight at 4 °C. The sections were washed twice with PBST (PBS with 1% tween) and once with PBS, and then incubated with secondary antibodies and DAPI mix for 1 h at room temperature. After washing for three times with PBS, the sections were covered with anti-florescence quencher and sealed with nail polish.
TUNEL staining was applied using the one-step TUNEL apoptosis assay kit according to the manufacturer's instructions. Briefly, the sections were dewaxed, rehydrated, and retrieved using antigen. Then, the sections were incubated with 20 μg/mL of protein K for 30 min at room temperature. After washing with PBS, the sections were incubated with 50 µL of TUNEL detection solution (5 µL of TdT enzyme and 45 µL of fluorescein labeling solution) at 37 °C for 1 h.

For Immunofluorescence, TNBC cells were seeded in the confocal dish at a density of 5 × 10⁵ cells per well. After 24 h, chemical regiments are added. The next day, cells were washed and fixed with 4% formaldehyde for 30 min at RT. Then cells were permeabilized with 1% Triton X-100 for 20 min. The non-specific binding sites were later blocked by blocking solution (Beyotime) for 2 h. The cells were incubated with specific primary antibodies overnight at 4 °C. After washing with PBS, the corresponding secondary antibodies conjugated to Alexa Fluor 555 and Alexa Fluor 488 were applied at room temperature for 1 h and then stained with DAPI (1:1000, Beyotime) for 5 min. The specimens were detected and imaged by a laser-scanning confocal microscope (Olympus, Japan).

The collected cells were lysed with lysis buffer-containing protease inhibitors (Beyotime, Shanghai, China) and placed in a pre-cooled centrifuge, and centrifuged at 13,000× g for 20 min. The BCA Protein Assay Kit (Beyotime, Shanghai, China) was then used to quantify the protein concentration. Next, 20 μg of total protein was separated by 12% polyacrylamide gel electrophoresis and transferred to a PVDF membrane by Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad, Hercules, CA, USA), and blocked with blocking solution (Beyotime, Shanghai, China) for 1 h at room temperature. After blocking, the membrane was placed at 4 °C to react with the primary antibody overnight and then washed 3 times with TBST and incubated with the secondary antibody for 1 h at room temperature. Finally, it was washed three times with TBST, and the BeyoECL Star Kit (Beyotime, Shanghai, China) was used to visualize the protein bands. According to the manufacturer’s protocol, the image is presented through Amersham Imager 600 (GE Healthcare Bio-Sciences Corp., Piscataway, NJ, USA). The primary antibody information used is as follows: anti-Flag (diluted 1:1000, Abclonal, Wuhan, China), anti-Tubulin (diluted 1:1000, GenScript, Nanjing, China), followed by the horseradish peroxidaseconjugate secondary antibody (diluted 1:10,000, GenScript, Nanjing, China).

Following the treatments described above, PC12 cells were washed twice with cold PBS and lysed with radio immunoprecipitation assay buffer (RIPA; P0013C, Beyotime Institute of Biotechnology, China). Cell lysates were centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected for further testing. The concentration of the protein samples were detected using a BCA kit (P0010S, Beyotime, China). Equal amounts of protein were separated by 10% or 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. After 30 min of blocking with the blocking solution (Beyotime Biotechnology), the membranes were incubated with primary antibodies overnight at 4 °C, followed by fluorescent secondary antibody (1:10000; Thermo Fisher, Invitrogen) for 1 h at room temperature. Then, the protein was visualized using a fluorescence imaging scanner (LI-COR, Odyssey CLx, US). β-actin was used as an internal reference, and protein expression is shown as the ratio of the band optical intensity to that of β-actin.