Vegfr1

The fresh transplanted tumor tissue was fixed in a 10% formalin fixed solution for 24 hours, dehydrated in gradient alcohol, and then the tissue blocks were placed transparently in xylene. The transparent tissue blocks were embedded in liquid paraffin, sliced after solidification, and baked on glass slides. Prior to staining, dehydration was performed with xylene and gradient alcohol dewaxing, and distilled water washing followed by inactivation of endogenous peroxidase with 3% hydrogen peroxide solution and antigen repair with sodium citrate buffer. After serum sealing, first antibody (caspase3, caspase8, Bcl-2, VEGF, VEGFR-1, PCNA, Ki-67, CD163, CD206, Fas, Survivin) (1:200)(all purchased from Abcam)was added to incubate overnight at 4°C and rinsed 3 times with PBS. Then we added second antibody: anti-rabbit polyclonal antibody (1:5000) or anti-mouse antibody (1:5000) polyclonal antibodies, incubated at 37°C for 30 minutes and rinsed 3 times. We dropped the chromogenic solution, hematoxylin dye re-dyeing, dehydration, transparent with xylene, and gum sealed microscopic examination.

The transfected cells were plated into 96-well plates at a density of 4000 cells/well. The cells were fixed with ice-cold methanol and blocked with serum. The primary antibodies of VEGFR1, VEGFR2, and VE–Cad (Abcam, USA) were used at a 1:100 work solution. Fluorescein isothiocyanate- and tetramethyl rhodamine isothiocyanate-conjugated mouse and rabbit IgG antibodies (Santa Cruz Biotechnology, USA) were used to label immunofluorescence. After immunolabeling, the cells were stained with Hoechst (Sigma, Germany) and measured with high-content screening (HCS) systems to evaluate the protein expression (Thermo Scientific, USA).

AAV9-CMV-spCas9-pA, AAV9-(H1-sgRNA(VEGFB)sp)×3-CAG-DIO-mCherry, AAV9-(H1-sgRNA(VEGFR1)sp)×3-CAG-DIO-mCherry, and AAV9-H1-sgRNA(NC)sp-CAG-DIO-mCherry were designed and validated by Shanghai Taitool Bioscience Co., Ltd. Antibodies against PCNA and Cyclin D1 were purchased from Zen-bioscience Company (Chengdu, Sichuan, China). Antibodies against VEGFB, VEGFR1, CD31, and MyoG were purchased from Abcam (Cambridge, MA, USA). Antibodies against VEGFR2, NRP1, and MyoD were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibody against MyHC was purchased from R&D systems (Minneapolis, MN, USA). Antibodies against PPARγ, C/EBPα, and FABP4 were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies against UCP1, CD36, FATP3, and FATP4 were purchased from Proteintech Group, Inc. (Rosemont, IL, USA). Antibodies against β-tubulin and GAPDH were purchased from Bioworld Technology, Co, Ltd. (Nanjing, China). The goat anti-mouse HRP-conjugated secondary antibody and goat anti-rabbit HRP-conjugated secondary antibody were purchased from Bioworld Technology, Inc. (St. Louis Park, MN, USA).

IHC was performed to detect the expression levels of different proteins. Tissue sections were deparaffinized in xylene and rehydrated by gradient alcohol prior to IHC. Endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide in methanol for 30 min. The tissue sections were heated using 0.01 M citric acid buffer for 10 min in a microwave oven. The slides were stained with antibodies against Twist1 (1:100, Santa Cruz Biotechnology), TP (1:100, Abcam), VE–cad (1:50, Abcam), VEGFR1 (1:250, Abcam), VEGFR2 (1:50, Abcam), or CD31 (1:50, Abcam). After incubation with horseradish peroxidase-conjugated goat anti-rabbit/mouse immunoglobulin G (IgG), the sections were stained with 3,3′-diaminobenzidine and counterstained with hematoxylin or periodic acid–Schiff (PAS). Finally, the sections were dehydrated and mounted. IHC staining was scored by multiplying the positive degree (0 for none, 1 for weak brown, 2 for moderate brown, and 3 for strong brown) and positive rate (1 for 0–25%, 2 for 25%–50%, 3 for 50%–75%, and 4 for 75%–100%). Sections with staining indices >6 were classified to the high-expression group. PAS-positive channels without CD31 staining were classified to the VM group. Five random ×40 fields per HCC tissue were scored with morphology consistent with mimicry. All quantification experiments were performed in a blinded setting.

The protein levels of Leptin-R, VEGFR-1, STAT3, p-STAT3, and BCL-2 after drug treatments were evaluated using Western immunoblotting. The total protein was extracted from the tumor tissues, and the total protein concentration was detected by the BCA Protein Concentration Assay Kit. Proteins were separated using SDS-PAGE and transferred to PVDF membranes. The membranes were blocked in 5% bovine serum albumin (BSA) for 2 h and then washed with TBST three times, each for 5 min on a shaker. The primary antibodies used were Leptin-R (Abcam 1:2000), STAT3 (CST 1:2,000), p-STAT3 (CST 1:1,000), BCL-2 (Abcam 1:2,000), VEGFR-1 (Abcam 1:2,000), and β-actin (Abcam 1:2,000). After incubation overnight at 4°C with primary antibodies, the membranes were washed with TBST and then incubated with one of the following secondary antibodies at room temperature for 1 h: HRP-labeled goat anti-rabbit secondary antibody (Jackson 1: 2,000) or HRP-labeled goat anti-mouse secondary antibody (Jackson 1:2,000). The immunoblots were then visualized using enhanced chemiluminescence (ECL) reagents and exposed to X-ray film. The membranes were subsequently re-probed with anti-β-actin antibody. Western blots were performed according to the manufacturer’s instructions with standards run in triplicate. The gray values of the immunoblots were semi-quantitatively determined by image analysis.

Immunofluorescence staining was conducted to evaluate the expression of
angiogenic proteins (CD31/VEGFR1) on the membrane of HUVECs. Briefly, after
placing one coverslip in each well of 6-well plate, 0.5 × 10⁴HUVECs were seeded and cultured in α-MEM medium. After 6 h when HUVECs
were attached on the coverslip, α-MEM medium was replaced by extracts.
Having been cultured in extracts for 3 days, HUVECs were rinsed twice by PBS and
were fixed by 4% paraformaldehyde for 20 min. Subsequently, CD31 (Abcam,
Cambridge, UK) and VEGFR1 (Abcam) antibodies were used to assess CD31/VEGFR1
expression. Photos were taken by the inverted fluorescence microscope (NIKON
ECLIPSE TI-SR, Tokyo, Japan).