Vascular endothelial growth factor (vegf)

Bev was obtained from Roche (Roche Pharma Ltd., Basel, Switzerland). An was offered by the Chiatai Tianqing pharmaceutical group (Tianqing Co., Nanjing, China). Dimethyl sulfoxide (DMSO) (Solarbio, Beijing, China) was used to dissolve An. Antibodies against VEGFR2, PDGFR, AKT, and p-AKT were purchased from Abcam (Cambridge, MA, USA); VEGF was purchased from Affinity (Cincinnati, OH, USA); and FGFR was purchased from Cell Signaling Technology (Danvers, MA, USA). The matrix gel was purchased from BD Bioscience (Pasadena, CA, USA). The Roswell Park Memorial Institute (RPMI) 1640 medium (Thermo Fisher Scientific, NYC, USA), Dulbecco's Modified Eagle's medium (DMEM) (Thermo Fisher Scientific), or Normal Saline (NS) was used for preparing working solutions from the stock solution in vitro or in vivo. The DMSO concentration in the final product was <0.1 %.

Western blotting analysis was performed according to our previous study⁸ (link)^,35 . Briefly, cell samples or mouse tumor tissues were lysed by RIPA lysis buffer containing protease, phenylmethanesulfonyl fluoride, and phosphatase inhibitors. Bicinchoninic acid (BCA) protein assay was carried out to determine the total protein concentrations. Next, proteins were separated by 10% SDS-polya-crylamide gel electrophoresis and the protein bands were further electro-transferred onto the PVDF membrane. Subsequently, the PVDF membranes were incubated at 4 °C with corresponding primary antibodies against Vinculin (1:1000, Affinity, Temecula, CA, USA), phosphoSTAT1 (1:1000, Affinity), total STAT1 (1:1000, Affinity), PD-L1 (1:1000, Affinity), β-actin (1:5000, Affinity), AMPK (1:1000, CST, Danvers, MA, USA), phospho-AMPK (Thr 172, 1:1000, CST), HIF-1α (1:1000, Affinity), CD31 (1:1000, Affinity), α-SMA (1:1000, Affinity), and VEGF (1:1000, Affinity) with gentle shaking overnight. 24 h later, the membranes were exposed to secondary antibodies at room temperature for another 2 h. Finally, the images of the protein bands were acquired by using the Fluorescent & Chemiluminescence Gel Imaging System (Peiqing Science and Technology Co., Ltd., Shanghai, China).

About 5×10⁶ cells were gathered after transfection for 48 h and lysed in RIPA buffer (Cell Signaling Technology, Danvers, MA, USA) in the presence of protease inhibitor (PMSF) and phosphatase inhibitor (Na-orthovanadate and NaF). The whole cell extracts were analyzed by SDS-PAGE and transferred to a nitrocellulose membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad). After incubation with 10% nonfat milk in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.1% Tween 20) for 1 h, the membrane was washed twice for 10 min with TBST and incubated with antibodies against p62 (1:1,000), LC3 (1:1,000), Beclin1 (1:2,000), RAGE (1:2,000), HMGB1 (1:1,000), VEGF (1:1,000), VEGFR2 (1:2,000), or β-actin (1:2,000, affinity biosciences) at 4°C overnight. Membranes were washed three times for 10 min and incubated with a 1:5,000 dilution of horseradish peroxidase-conjugated anti-mouse or anti-rabbit antibodies for 1 h. Blots were washed with TBST 3 times for 10 min and developed with the ECL system (Millipore, Darmstadt, Germany) according to the manufacturer’s protocols. Results were normalized to the internal control β-actin.