Clinical samples

Serum samples were collected from 50 patients with atherosclerosis from Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital. Meanwhile, 50 serum samples were obtained from healthy volunteers and serve as normal control. The diagnosis of atherosclerosis was based on the value of the carotid intima-media thickness (CIMT) of the common carotid artery. Exclusion criteria were cardiovascular and cerebrovascular diseases, cancer, or infection. The blood samples were taken before any medications were given to the patients. Before sample collection, written informed consent was provided by all participants. Table 1 shows the clinical characteristics of the patients, and their association with atherosclerosis. This study was approved by the Ethics Committee of Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital.

Table 1 Clinical characteristics of subjects in the studyAnimal model

Six-week-old male ApoE–/– mice were purchased from Hunan SJA Co., Ltd (Changsha, China) and fed with a Western high fat diet (Hunan SJA Co., Ltd) for 12 weeks. For inactivation of Smad2/3 pathway, 4.2 mg/kg SB431542 (MedChemExpress, USA) was daily intraperitoneally injected into mice. DMSO was served as the vehicle control. Finally, all mice were sacrificed by cervical dislocation and the aortic tissues were obtained. The animal protocol was approved by the Ethics Committee of Zhengzhou University People’s Hospital, Henan Provincial People’s Hospital.

Cell culture and treatment

Human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (USA) and cultured in endothelial growth medium (ScienCell, USA) supplemented with 5% FBS (Gibco, USA) and endothelial growth factors. The confluent HUVECs at passages 6–7 were maintained in basal medium containing 0.5% FBS for 8 h, followed by stimulation with ox-LDL (100 µg/mL) for 24 h to induce EndMT (Diao et al. 2022; Gong et al. 2019; Qiu et al. 2016). Thereafter, the cells were collected for further experiments.

Cell transfection

HUVECs were transfected with shRNA targeting USF1 (sh-USF1), sh-USP14, USF1 overexpression plasmid (oe-USF1), oe-USP14, oe-NLRC5, or corresponding negative controls (sh-NC, oe-NC) using Lipofectamine 2000 (Thermo Fisher, USA). All shRNAs or plasmids were provided by GenePharma.

Quantitative real time polymerase chain reaction (RT-qPCR)

Total RNA was isolated from human serum samples, HUVECs, and aortas using TRIzol reagent (Thermo Fisher) and reverse-transcribed into cDNA using the RT Master Mix for qPCR II (MCE, USA). RT-qPCR was performed using the Super Real Premix Plus Kit (Tiangen, Beijing, China). The levels of genes normalized to GAPDH were calculated using the 2−ΔΔCT method. Table 2 shows the primer sequences.

Table 2 Oligonucleotide primer sets for RT-qPCRWestern blotting

Protein samples were extracted using the commercial extraction kit (Thermo Fisher). The protein lysates were subjected to SDS-PAGE and blotted onto polyvinylidene fluoride membranes. After blocking in 5% bovine serum albumin for 1 h, the membranes were incubated with the primary antibodies against CD31 (A19014, 1:1000, ABclonal, Wuhan, China), VE-Cadherin (bs-22363R, 1:500, Bioss, Beijing, China), α-SMA (A17910, 1:500, ABclonal), vimentin (A19607, 1:1000, ABclonal, Wuhan, China), USF1 (sc-390,027, 1:1000, Santa Cruz, USA), USP14 (A19589, 1:500, ABclonal), NLRC5 (sc-515,668, 1:1000, Santa Cruz), Ubiquitin (ab140601, 1:1000, Abcam, UK), Smad2 (A19114, 1:500, ABclonal), Smad3 (A19115, 1:500, ABclonal), p-Smad2 (AP0269, 1:500, ABclonal), p-Smad3 (AP0727, 1:500, ABclonal), β-actin (AC006, 1:1000, ABclonal) overnight at 4 °C. Subsequently, the membranes were reacted with the secondary antibody (1:5000, ABclonal). The membranes were visualized using the Clarity Western ECL Substrate (BIO-RAD, USA).

Immunofluorescence staining

The aortic tissues were fixed with 4% paraformaldehyde and cut into 4-µm sections, followed by permeabilization with 1% Triton X-100 and blocking with 5% BSA. For HUVECs, the cells with various treatments were fixed with 4% paraformaldehyde, infiltrated with 1% Triton X-100, and sealed with 5% BSA. Subsequently, the aortic sections or HUVECs were incubated with primary antibodies against CD31 (A19014, 1:50, ABclonal), α-SMA (A17910, 1:50, ABclonal) overnight at 4 °C. Then, FITC Goat Anti-Rabbit IgG (AS011, ABclonal) or Cy3 Goat Anti-Rabbit IgG (AS007, ABclonal) was applied. Finally, the sections or HUVECs were stained with DAPI, examined under a fluorescence microscope (Olympus, Japan) and quantified using the Image J software.

Histopathological analysis

The collected aortic tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 4 μm-sections. Subsequently, the sections were stained using the HE Staining Kit (Solarbio, Beijing, China) and Masson’s Trichrome Stain Kit (Solarbio) to evaluate atherosclerotic plaque formation and collagen accumulation, respectively. The sections were photographed under a light microscope.

Enzyme linked immunosorbent assay (ELISA)

Serum samples were collected from the peripheral blood of mice. The cellular supernatant of HUVECs was collected. TNF-α, IL-1β, IL-6 levels in serum samples and cellular supernatant were assessed by ELISA using the commercial kits provided by Solarbio, according to the manufacturer’s instructions.

Transwell assay

HUVECs were suspended in serum-free medium (2 × 105 cells/mL). The upper chambers were added with 100 µL of cell suspension, while the bottom chambers were added with 800 µL of medium containing 10% FBS. After maintenance for 12 h, the unmigrated cells on upper chambers were wiped off. The migrated cells were fixed with methanol and stained with crystal violet. The photographs were taken and quantified using Image J software.

Scratch assay

HUVECs were seeded into 12-well plates and cultured until confluence. A scratch was made using a pipette tip (500 µL), followed by washing with serum-free medium. At 0 and 48 h after scratch, HUVECs were photographed under a light microscope.

Dual-luciferase reporter assay

The promoter sequences of USP14 were amplified and inserted into the pGL3 vector, named as wt-USP14. Mutant construct mut-USP14 was established by mutagenesis. The above luciferase reporter plasmids together with oe-USF1 or oe-NC were co-transfected into HUVECs using Lipofectamine 2000. The relative luciferase activity was detected using the Dual-Lucy Assay Kit (Solarbio) at 48 h after transfection.

Co-immunoprecipitation (Co-IP)

HUVECs were lysed with the lysis buffer for IP (Beyotime, China) supplemented with PMSF and cocktail. Cell lysates were incubated with Protein A + G agarose beads at 4 °C for 1 h, followed by incubation with primary antibody against NLRC5 (sc-515,668, Santa Cruz) that was pre-conjugated in Protein A + G agarose beads overnight at 4 °C with rotation. Thereafter, the protein samples were eluted and determined by Western blotting.

Chromatin immunoprecipitation (ChIP)

ChIP assay was performed using the ChIP kit (Abcam) following the manufacturer’s protocol. Briefly, HUVECs were cross-linked using 1% formaldehyde and then treated with glycine. Subsequently, ultrasonication was performed to break the chromatin. The chromatin fragments were precipitated with anti-IgG or anti-USF1 (Santa Cruz) at 4 °C. After treatment with NaCl and protease K, the DNA was purified and the enrichment of USP14 was measured by RT-qPCR.

Oil red O staining

Plaque formation was evaluated by oil red O staining. Briefly, the aortic tissues were fixed in 4% paraformaldehyde and dehydrated with 15% and 30% sucrose, followed by embedding with optimal cutting temperature glue. The aortic tissues were sliced into 8-µm-slices and stained with Oil Red O solution (Solarbio) at 37 °C for 2 h. Under a light microscope, the images were taken and the plaque area was quantified according a previous study (Jiang et al. 2019).

Verhoeff-Van Gieson (EVG) staining

To determine morphological changes, the sections of aortic tissues were deparaffinized and hydrated. The sections were immersed in EVG solution for 30 min, followed by incubation with ferric chloride differentiation solution until the background was grey white. Then, the sections were re-stained with VG solution. After dehydration using 100% ethanol, the sections were photographed under a light microscope.

Statistical analysis

All values are expressed as mean ± standard deviation (SD) and analyzed by GraphPad Prism 8.0 software. For in vitro assay, both technical replicates and biological replicates were performed. For in vivo assay, biological replicates were performed. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test or Student’s t test wase performed to evaluate the differences between two groups or multiple groups. Correlation between USF1 and USP14 expression was analyzed by Pearson correlation analysis. P < 0.05 was considered statistically significant.