Vegfa

DEHP (Sigma-Aldrich, 36735, Saint Louis, MO, USA) was reconstituted in DMSO (Sigma-Aldrich, Saint Louis, MO, USA). Doxorubicin (Dox) (Sigma-Aldrich, D1515, Saint Louis, MO, USA) was dissolved in DMSO (Sigma-Aldrich, Saint Louis, MO, USA). Primary antibodies for proteins: Endoglin/CD105 (71 kDa, Proteintech, 10862-1-AP, Rosemont, IL, USA), MAPK (ERK1/2) (44 kDa, Cell Signaling, 137F5, Danvers, MA, USA), phospho-MAPK (ERK1/2) (Thr202/Tyr204) (44 kDa, Cell Signaling, 9101 s, Danvers, MA, USA), p38 (38 kDa, Proteintech, 14064-1-AP, Rosemont, IL, USA), phospho-p38 (Tyr182) (38 kDa, Santa Cruz, SC-166182, Santa Cruz, CA, USA), GAPDH (35.5 kDa, EMD Millipore, MAB374, Burlington, MA, USA), Smad2/3 (52, 60 kDa, Cell Signaling, 8685, Danvers, MA, USA), phospho-Smad2 (Ser465/467)/Smad3 (Ser423/425) (52, 60 kDa, Cell Signaling, 8828, Danvers, MA, USA), β actin (43 kDa, Santa Cruz, SC-47778, Santa Cruz, CA, USA), and VEGFA (43 kDa, Proteintech, 66828-1-IG, Rosemont, IL, USA). Secondary antibodies: Goat anti-rabbit IgG (Alexa Fluor 594, Invitrogen, A11012, Waltham, MA, USA) was used as the secondary antibody.

Total cell lysates were separated by SDS‐PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and blotted on polyvinylidene difluoride membranes (Millipore). Then, the membranes were blocked with 5% milk (170‐6404, Bio‐Rad Laboratories, Inc) and incubated with specific antibodies to Klf3 (Invitrogen Antibodies, PA5‐18030, 1:1000), JunB (Cell Signaling Technology, 3753, 1:1000), VEGFA (Proteintech, 19003‐1‐AP, 1:1000) and α‐Tublin (Proteintech, 11224‐1‐AP, 1:2000). Blots were visualized using SuperSignal West Pico PLUS Chemiluminescent Substrate (SD251210, Thermo Fisher Scientific, Inc).

After the MDA-MB-231 cells were treated with XLLXF (50, 100, and 200 µg/mL) for 24 and 48 h, total cell protein lysates were extracted using RIPA lysis buffer that contained protease and phosphatase inhibitor cocktails. Protein lysates (20 µg), which were determined by BCA analysis (Beyotime, China), were loaded onto 10% SDS-PAGE gels. The protein bands were transferred onto NC membranes and blocked with 5% non-fat milk for 1 h at room temperature. The NC membranes with proteins were incubated with diluted primary antibodies at 4 °C overnight. The primary antibodies used in the analyses were as follows: VEGFA (1:1,000, Proteintech), MMP2 (1:1000, Proteintech), MMP9 (1:1000, Cell Signaling Technology), Vimentin (1:1000, Proteintech), VE-cadherin (1:1,000, Cell Signaling Technology), TIMP-1 (1:1000, Proteintech), TIMP-3 (1:1,000, Proteintech), and Twist1 (1:1000, Proteintech). The membranes were incubated with relative sources of secondary antibodies (1:5000) at room temperature for 1 h. Specific protein bands were recognized with Immobilon Western Chemiluminescent HRP Substrate (Millipore, MA, USA). Image J software was used for image analysis.

The tissues were fixed in a 4% paraformaldehyde solution and embedded in paraffin. The tissue sections were dewaxed in xylene,
hydrated in graded alcohol solution, and heated (100 °C) in citric acid solution (pH 6.0) for antigen repair. For single
immunostaining, the endogenous peroxidase in the tissue was blocked using endogenous peroxidase blocking solution,
and 5% BSA was used to block the tissue’s nonspecific proteins. The sections were incubated overnight at 4 °C
with the indicated antibodies, followed by incubation with the corresponding horseradish peroxidase-coupled secondary
antibodies (#PV9001, Beyotime). Finally, 3,3′-diaminobenzidine (#PV6000D, Beyotime) was used to detect these labeled
antibodies and the nuclei were stained using hematoxylin. The primary antibodies were as follows: SHMT2
(1:100, #33443, Cell Signaling Technolog), HIF1α (1:100, #36169, Cell Signaling Technology), VEGFA (1:100, #19003, Proteintech),
KI67 (1:50, #5032, PTM Bio, Hangzhou, China), and CD31 (1:50, #ab28364, Abcam, Cambridge, MA, USA).

Rabbit anti-human MPP6, VEGFA and VEGFR2 polyclonal antibodies were provided from Proteintech (Wuhan, China), and anti-CD34 monoclonal antibody, anti-CD3+/CD4+/CD8+ T-cell monoclonal antibodies, secondary antibodies, and SP kits were purchased from Fuzhou Maishin Biotechnology (Fuzhou, China). These tissues were routinely dewaxed, hydrated, subjected to antigen repair with citrate buffer solution, and then assessed. All operating procedures strictly adhered to kit instructions. The MPP6, VEGFA and VEGFR2 primary antibody dilation ratio was 1:200, and anti-CD34 monoclonal antibody and anti-CD3+/CD4+/CD8+ T-cell monoclonal antibodies were ready-to-use antibodies. According to the positive signal, staining intensity was classified by unstained as negative, light yellow as (+), yellowish-brown as (++) and brown as (+++). Samples with MPP6 staining intensities of (+) and (+++) were chosen for VEGFA, VEGFR2 and CD34 staining to observe angiogenesis and CD3+/CD4+/CD8+ T-cell staining to evaluate immune cell distribution.

Proteins from cells were extracted with RIPA lysis buffer (Beyotime, China) supplemented with protease inhibitors (Beyotime, China). Proteins were separated through sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and then bolted on polyvinylidene difluoride (PVDF) membranes (Millipore, USA). After blocking nonspecific sites with 5% milk (Beyotime, China) for 1 hour, the PVDF membranes were incubated with primary antibodies against CD31, VEGFA, N-cadherin and vimentin (Proteintech, China) at 4 °C overnight. The membranes were then incubated with secondary antibodies (Bioss, China) at room temperature for 1 hour. The protein bands were visualized using a Fusion FX System (Vilber, Germany).