Cell culture

HUVECs (iCell-h110) were obtained from iCell Bioscience Inc, Shanghai. HUVECs were cultured in culture medium (iCell-h110-001b). Cells were passaged every 4 days with 0.25% trypsin. Medium was changed daily. Cells were routinely checked for normal morphology, the bacterial or mycoplasma contamination. Passages 3–10 of HUVECs were used for assay.

Lentivirus plasmid construction and package

The END1 sequence and its shRNA sequence were respectively cloned into pSLenti-CMV-END1-3xFLAG-PGK-Puro-WPRE and pCLenti-U6-shRNA1-CMV-Puro-WPRE. The interference sequences are listed in Supplementary Table 1. The virus packaging was performed using the packaging kit from SBI (Mountain View, CA, USA) and according to the manufacturer’s instructions. The virus particles were harvested and concentrated using PEG-it virus precipitation solution (SBI). Virus titer was determined in 293TN cells using the Global UltraRapid Lentiviral Titer Kit (SBI), according to the manufacturer’s instructions. HUVECs were infected with lenti-END1, lenti-sh-END1 and their Lenti-control, respectively, at a different multiplicity of infection in the presence of Polybrene (5 µg/mL; Sigma).

CCK8 assay

Collect cultured HUVECs and adjust cell concentration to 5 × 104 cells/mL, then 100 µL/well of cell suspension from different groups was inoculated in a 96-well plate. Pre-incubate the plate in a humidified incubator at 0 h, 24 h, 72 h and 120 h. In drug treating group, 10 µM BQ123 was added. Add 10 µl of the Cell Counting Kit-8 solution to each well of the plate. Incubate the plate for 2 h in the incubator. Measure the absorbance at 450 nm using a microplate reader.

Cell invasion assay

Human umbilical vein endothelial cells (HUVECs) were harvested and washed with 3 ml PBS. The cells were digested and collected with 0.25% trypsin, centrifuged at 1000 rpm for 5 min, and rinsed twice with PBS. The cells were re-suspended in serum-free medium and counted by cell counting plate. The cells were diluted to 3 × 105/ml in serum-free medium. The Matrigel was melted at 4 °C one day earlier, and the Transwell Chamber, 24-well culture plate and gun tip were precooled at -20 °C overnight. Matrigel was diluted to the final concentration of 1 mg/ml in serum-free medium, and operated on ice. 800 µl 10% FB medium was precooled at 4 °C in 24-well plates and placed in a transwell chamber. 100 ΜL Matrigel with a final concentration of 1 mg/ml was added vertically at the bottom of the upper chamber of the Transwell Chamber. Matrigel was incubated at 37 °C for 4–5 h to make it dry and gelatinous. After Matrigel was dried and gelatinous, 200 µl cell suspensions of each group were placed in the Transwell Chamber and cultured at 37 °C and 5% CO2 for 72 h in the chamber. Transwell was removed, the chamber was carefully washed with PBS, and the cells were fixed with 70% ice-ethanol solution for 1 h. The cells were stained with 0.5% crystal violet solution, placed at room temperature for 20 min, washed with PBS, and wiped with a clean cotton ball to clean the non-migrated cells on one side of the upper chamber and the other side.

Cell apoptosis

The HUVECs from different groups were collected, the trypsin was digested with EDTA-free medium and collected at room temperature at 1000 rpm for 5 min, collect cells. Cells were resuspended once with precooled 1xPBS (4 °C), centrifuged at 1000 rpm for 5 min, and washed. Add 300 ΜL of 1x Binding Buffer suspension cells. Annexin V-FITC was mixed with 5 µl of Annexin V-FITC and incubated at room temperature for 15 min. After adding 10 µl PI staining, the cells were incubated at room temperature for 10 min in the dark. Flow cytometry was used to test and analyze results.

RNA extraction and quantitative real-time polymerase chain reaction

Total RNA was extracted from the cultured cells using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. The extracted RNA was quantified using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Bremen, Germany). cDNAs were synthesized from the mRNA using TIANScript RT (Tiangen Biotech Co., Ltd, Beijing, China), with oligo (dT) primers used for reverse transcription. For qRT-PCR, the 7500 System (ABI) was utilized. SYBR Premix (F-415XL, Thermo) was used for the qRT-PCR reactions. The specific forward and reverse primers employed for reverse transcription and qRT-PCR are listed in Supplementary Table 2. All reactions were performed in triplicate. The relative mRNA expression levels were determined using the 2^(-ΔΔCt) method and normalized to the expression levels of the endogenous reference gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Western blot analysis

For Western blot analysis, total protein was extracted from the cultured cells using lysis buffer. 40 µg of the protein sample were separated by SDS polyacrylamide gel electrophoresis and subsequently transferred onto nitrocellulose membranes. The membranes were then incubated overnight at 4 °C with primary antibodies targeting specific proteins, including NQO1 (1:1000, ab80588, abcam), GCLC (1:1000, ab20777, abcam), Nrf2 (1:1000, ab62352, abcam). To ensure accuracy and consistency, the anti-GAPDH antibody (1:5000, 60004-1-Ig, Proteintech) was included as a loading control in each individual experiment. After three washes, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:7000, ZB2305, Zhongshanjinqiao Biotechnology Co., Ltd) for 1 h at room temperature. The antigen-antibody complexes were detected using enhanced chemiluminescence, and the resulting images were analyzed using a Chemiscope 5300 Pro (CLINX).

H&E staining

The inferior vena cava thrombosis tissues of rat were isolated and fixed in 4% PFA solution for two days, sequentially dehydrated with 70%, 95% and 100% ethanol, and defatted with xylene for 2 h before being embedded in paraffin. The 10-µm-thick section was cut and subjected to hematoxylin and eosin staining (H&E staining). The slices were observed under optical microscopy.

Enzyme-linked immunosorbent assay (ELISA)

Cell culture supernatant and plasma concentrations of ET-1 (D711033) and coagulation factor VII (MM-50735H2) were quantified using commercially available enzyme-linked immunosorbent assay (ELISA) kits. The detection range of ET-1 kit was from 5 pg/mL to 160 pg/mL, and the lowest detection concentration was less than 1.0 pg/mL. The detection range of coagulation factor VII kit was from 1.5625 ng/mL to 50 ng/mL, and the lowest detection concentration was less than 0.1 ng/mL. The measurements were performed in accordance with the manufacturers’ instructions, and each sample was assessed in triplicate to ensure accuracy. The absorbances at 450 nm were recorded, and the mean concentrations were calculated based on these measurements.

Rat model of deep venous thrombosis (DVT) using inferior vena cava stenosis

Under anesthesia, the rats’ intestine and mesentery were repositioned to the right side, followed by the surgical opening of the retroperitoneal cavity. Subsequently, the junction of the left renal vein and the inferior vena cava (IVC), along with the segment of the IVC located 1 cm below it, were meticulously separated. A number 0 silk ligature was then carefully passed around the left renal vein, securing both ends of the ligature through the right side of the abdominal wall, thereby facilitating extrusion from the abdominal cavity. Similarly, a ligature was applied to the common iliac vein. The bowel and its associated mesentery were subsequently reintegrated into the abdominal cavity, ensuring appropriate adjustment of the ligature position to prevent bowel compression. The two ligature lines were individually secured onto the abdominal wall, and 2 ml of normal saline was administered into the abdominal cavity to conclude the abdominal closure. Attention was paid to the tension of the ligature; inadequate tightness may result in insufficient blood flow blockade and modeling failure, whereas excessive tightness may induce abdominal wall retraction and intestinal compression, leading to adverse outcomes. The adequacy of ligature tension was calibrated to permit passage of a standard 2B pencil. Postoperatively, the animals were maintained under appropriate warmth and provided unrestricted access to food and water. After excising the IVC, thrombi can be measured and then snap frozen or formalin-fixed for further analysis. For BQ-123 group (3 mg/kg), it was delivered into DVT rat model by tail vein injection.

Statistical analysis

The data are presented as the mean ± standard deviation (SD) from three independent experiments. We used an unpaired t-test to compare two groups and one-way ANOVA to compare more than two groups. A sample size of 3 was selected so that at a significance level of 0.05 there at least 95% chance of detecting two SD’s difference in outcome between the groups. All data were processed using GraphPad Prism 8 (GraphPad Software, Inc., La Jolla, CA). All analyses were conducted using SAS software (version 9.3, 2002–2012; SAS Institute Inc., Cary, NC, USA). Statistical significance was defined as P < 0.05 for all comparisons.