Cell culture and transfection

HUVECs were obtained from Wuhan Punosai Life Technology Co., LTD (#CL-0122, Wuhan, China). HUVECs were cultured in endothelial cell medium (ECM, #SC1001-2, ScienCell, San Diego, CA) containing 10% fetal bovine serum (FBS, #16,140,071, Grand Island, NY) and 1% penicillin/streptomycin (#15,070,063, Gibco, Grand Island, NY), and incubated an atmosphere with 5% CO2 at 37 °C. HUVECs were cultured until they approached or reached a confluent quiescent state (HUVECs enter the quiescent phase when they reach 80–90% confluence, which was closer to the physiological environment of stable vascular endothelium). To construct an AS cell model, HUVECs were exposed to 100 μg/ml ox-LDL (#IO1300, Solarbio, Beijing, China) (Zhu et al. 2024; Hu et al. 2025). Untreated HUVECs were used as control.

The Trim28 overexpression (oe-Trim28) and p53 overexpression (oe-P53) sequences were obtained from NCBI website. The shRNA targeting ALDH4A1 (sh-ALDH4A1) sequences (sense sequence: ACTTCTACATCAACGACAA and antisense sequence: TTGTCGTTGATGTAGAAGT) were obtained in the Designer of Small Interfering RNA website. The oe-Trim28, oe-P53, sh-ALDH4A1 and their negative control (NC) were integrated into pLKO.1 lentiviral vector to construct their lentiviral vectors. To generate high-titer lentivirus, the lentiviral vectors together with pMDLg/pRRE:pVSV-G:pRSV-Rev (5:3:2) packaging plasmids were cotransfected into 293 T cells using Lipofectamine™ 2000 Transfection Reagent (#11,668,030, Invitrogen, Carlsbad, CA). The titer of oe-Trim28 lentivirus was measured to be 7 × 108 TU/mL, with a multiplicity of infection (MOI) of 10. The titer of oe-P53 lentivirus was measured to be 7.4 × 108 TU/mL, with a MOI of 20. The titer of sh-ALDH4A1 lentivirus was measured to be 8 × 108 TU/mL, with a MOI of 10. The ox-LDL-treated HUVECs were inoculated into a 6-well plate, with 2 × 105 cells per well, and cultured until the cell density reach 70–90%. Cells were then infected with oe-Trim28 lentivirals, sh-ALDH4A1 lentivirus, or/and oe-P53 lentivirus using Lipofectamine™ 2000 Transfection Reagent. After 48 h of transfection, cells were harvested.

Cell line authentication

Authentication testing of HUVECs have been performed by Shanghai Biowing Biotechnology Co., Ltd (Shanghai, China) via STR profiling. DNA was extracted by a commercial kit from CORNING (#AP-EMN-BL-GDNA-250G, Corning, NY). The twenty STRs including Amelogenin locus were amplified by six multiplex PCR and separated on ABI 3730XL Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA). The signals were then analyzed by the software GeneMapper. Mycoplasma detection of HUVECs have been performed by Shanghai Biowing Biotechnology Co., Ltd (Shanghai, China). All the experiments in this study were performed with mycoplasma-free cells.

CCK-8 assay

A CCK-8 kit (#C0037, Beyotime, Jiangsu, China) was used for evaluating cell viability. HUVECs were seeded in a 96-well plate (2000 cells/well) and cultured at 37℃ with 5% CO2. After 24 hours treatment, each well was incubated with CCK-8 regent for 2 h. Absorbance at 450 nm was determined by a microplate reader (DR-3518G, Hiwell-Diatek Instruments Co., Ltd, Wuxi, China).

Oil red O staining

Oil red O staining kit (#C01578, Beyotime, Jiangsu, China) was conducted to detect intracellular lipid accumulation. HUVECs were treated with 4% paraformaldehyde for 10 min for fixation, and stained with oil red O solution for 30 min. After staining, the plates were observed under a microscope (DMi3000 B, Leica, Wetzlar, Germany).

Detection of total cholesterol (TC) and triglyceride (TG)

After cell supernatants collection, the TC and TG concentrations in cell supernatants were detected using the TC detection kit (#E-BC-K109-M2, Elabscience, Wuhan, China) and TG detection kit (#E-BC-K261-M1, Elabscience, Wuhan, China).

Western blotting

Total protein was harvested using RIPA lysis buffer (#P0013B, Beyotime, Jiangsu, China). Protein concentrations were determined by a BCA kit (#P0010S, Beyotime, Jiangsu, China). The protein was isolated with a 10% SDS-PAGE and transferred onto PVDF membranes (#FFP24, Beyotime, Jiangsu, China). After sealed with non-fat milk, the membranes were incubated overnight at 37 °C with ALDH4A1 rabbit polyclonal antibody (#11,604–1-AP, 1: 1000, Proteinech, Wuhan, China), Trim28 rabbit polyclonal antibody (#PA5-27,648, 1: 1000, Thermo, Waltham, MA), recombinant anti-P53 rabbit monoclonal antibody (#ab32509, 1: 1000, Abcam, Cambridge, MA), SLC7A11/Xct rabbit polyclonal antibody (#26,864–1-AP, 1: 1000, Proteinech, Wuhan, China), GPX4 polyclonal antibody (#30,388–1-AP, 1: 1000, Proteinech, Wuhan, China), and GAPDH rabbit monoclonal antibody (#ab181602, 1: 1000, Abcam, Cambridge, MA), followed by incubation with secondary goat anti-rabbit IgG H&L (HRP) antibody (#ab6721, 1: 10,000, Abcam, Cambridge, MA) for 1 h. The proteins were visualized with an ECL chemiluminescent reagent (#P1000, Puli Lai Gene Technology Co., Ltd., Beijing, China) and quantified.

Ubiquitination assay

The ox-LDL-treated HUVECs were first transfected with oe-Trim28, sh-ALDH4A1, or their corresponding NC. The cells were then collected for co-immunoprecipitation (co-IP) using a recombinant Anti-P53 Antibody (#ab32509, 1: 30, Abcam, Cambridge, MA), followed by immunoblotting using recombinant Anti-Ubiquitin rabbit monoclonal antibody (#ab134953, 1: 1000, Abcam, Cambridge, MA) to detect ubiquitin. The proteins were visualized with an ECL chemiluminescent reagent (#P1000, Puli Lai Gene Technology Co., Ltd., Beijing, China) and quantified.

Co-IP assay

HUVECs were lysed on ice for 5 min using pre-cooled Lysis/Wash Buffer. The protein supernatants were collected for co-immunoprecipitation using the IP/co-IP kit (#abs9649, Absin, Shanghai, China). In brief, the rProtein A/G MagPoly Beads were incubated with anti-ALDH4A1 or anti-Trim28 antibodies for 30 min. Afterwards, cell lysate samples were added to incubate with Beads. The precipitates were incubated with IP Elution Buffer for 10 min. After incubating Neutralization Buffer, the elution fraction was collected for western blotting.

Ferrous iron assay

HUVECs (1 × 105 cells/well) were seeded in 96-well plate. An iron assay kit (#MAK025, Sigma, St. Louis, MO) was applied to determine the intracellular ferrous iron (Fe2+) levels in HUVECs.

Molecular docking

The crystal structures of ALDH4A1 (3v9g) and Trim28 (2yvr) proteins were obtained from the UniProt database. Then, molecular docking was conducted using the Gramm online tool (https://gramm.compbio.ku.edu/request) (Singh et al. 2024). Finally, the docking results were imported into pymol software for visualization.

Statistical analysis

All experimental data (means ± SD) were analyzed with GraphPad 8.0. Student's t tests were used to analyze differences between the two groups, and one-way analysis of variance assay for multigroup analysis. A value of P < 0.05 indicated statistically significant results.