2.1 Increased 5-hmC Levels in Senescent Endothelial Cells and PQ-Induced Aging Mice

This study aimed to identify the phenotype of senescent endothelial cells by examining various senescence-related markers in human umbilical vein endothelial cells (HUVECs). The proportion of cells exhibiting positive β-galactosidase staining was greater in replicative senescent cells compared with young cells (Figure 1A). Additionally, the mRNA and protein levels of senescence-related markers p16, p21, and p53 were higher in the replicative senescent cells than in the young cells (Figure 1B; Figure S1). In comparison to young cells, replicative senescent cells exhibited elevated levels of several genes associated with inflammatory cytokines, cardiovascular function, and epigenetic regulation (Figure S1). However, the expression levels of certain critical genes, such as nitric oxide synthase 3, superoxide dismutase 2, sirtuin 3, and lysine methyltransferase 2D, were reduced (Figure S1). Dot blot analysis demonstrated a significant increase in the density of 5-hmC in replicative senescent cells compared with young cells (Figure 1C). Immunofluorescence analysis further revealed that replicative senescent cells exhibited higher intensities of 5-hmC and phosphorylated H2A.X variant histone (p-γH2AX), a marker of DNA damage, relative to young cells (Figure 1D). To further investigate the alterations in 5-hmC within senescent cells, PQ was employed to induce senescence in HUVECs. Following treatment with 20 µM PQ for 3 and 4 days, there was a significant upregulation in the mRNA levels of p16, p21, and p53 (Figure S2A). Additionally, both the number of cells positively stained for β-galactosidase and the protein levels of p16, p21, and p53 were elevated after 3 days of 20 µM PQ treatment (Figure S2B,C). In addition, PQ also significantly induced senescence in H9C2 cells, human embryonic kidney (HEK) 293T cells, human kidney-2 (HK2) cells, and human aortic vascular smooth muscle cells (T/G HA) cells (Figure S2D–G). Furthermore, cell cycle analysis revealed that PQ markedly increased the proportion of cells in the G2/M phase while decreasing the proportion of cells in the S phase (Figure S2H). These findings indicate that PQ is a potent inducer of cellular senescence.

To investigate the alterations of 5-hmC in PQ-induced aging mice, the animals were administered PQ intraperitoneally at a dosage of 10 mg/kg per week for 4 weeks to induce aging. The results demonstrated significant upregulation of mRNA levels of senescence-associated markers p16, p21, and p53 in the heart, kidney, and aorta of PQ-treated mice (Figure S3A–E). PQ treatment resulted in increased mRNA expression of inflammatory cytokines in the heart (Figure S3F,G), and induced myocardial fibrosis (Figure S3H). Additionally, levels of phosphorylated p-γH2AX and 5-hmC were significantly elevated in the heart, kidney, and aorta of PQ-treated mice (Figure 1E,F; Figure S3I). However, PQ treatment did not induce apoptosis in the heart and kidney tissues of PQ-induced aging mice (Figure S3J). These results indicated that 5-hmC levels were markedly elevated in senescent cells and cardiovascular tissues of PQ-induced aging mice.

To explore the alterations in DNA methylcytosine (5-mC) and 5-hmC levels in replicative senescent HUVECs, methylated DNA immunoprecipitation sequencing (MeDIP-seq) and hydromethylated DNA immunoprecipitation sequencing (hMeDIP-seq) were investigated. Bioinformatic analysis of the sequencing data revealed significant changes in DNA 5-hmC levels compared with 5-mC levels between the replicative senescent cells and the young cells. Specifically, a greater increase in the levels of 5-hmC was found in the replicative senescent cells than in the young cells (Figure S4A, Tables S1, S2). The levels of 5-mC were relatively unchanged changed in p21, and p53, whereas the levels of 5-hmC were substantially increased (Figure 1G,H; Figure S4B). Analysis of the gene ontology (GO) showed that at the biological phenotype level, 5-hmC was involved in the induction of nitric oxide synthase activity, cholesterol catabolism, negative regulation of endothelial cell migration, histone lysine demethylation, and positive regulation of protein complex disassembly, whereas at the molecular function level, it was involved the activities of numerous enzymes, such as histone demethylase, and methyltransferase as well as in the activities of translation repressor, intracellular ligand-gated calcium channel, and phospholipid-translocating ATPase in replicative senescent endothelial cells (Figure S4C). These results indicated that replicative senescent endothelial cells had a higher susceptibility to changes in genomic 5-hmC, but not 5mC.

2.2 TET3 controls the Cellular Senescence Phenotype

While some studies have suggested that the levels of TET2, and TET3 fluctuate with aging, either increasing or decreasing, definitive genetic evidence remains lacking [15, 33, 34]. This study aimed to elucidate the role of TET2 and TET3 in cellular senescence by examining their levels in senescent endothelial. The mRNA and protein levels of TET2 and TET3 were higher in the replicative senescent HUVECs compared with the young HUVECs, as well as in the PQ group compared with the control group (Figure 2A,B; Figure S5A,B). In addition, PQ treatment markedly increased the mRNA levels of TET2 and TET3 in HEK293T, and HK2 cells, and also markedly increased the mRNA level of TET3 in H9C2, and T/G HA cells (Figure S5C–F). Previous research has demonstrated that STAT3 can act as a transcription factor to regulate the expression of TET3 [35]. To investigate whether PQ modulates TET3 expression via STAT3, Western blotting and chromatin immunoprecipitation (ChIP) assays were conducted. The results revealed that PQ significantly enhances the phosphorylation of STAT3 and its binding to the TET3 promoter (Figure S5G,H). The knockdown of TET3, as opposed to TET2, effectively inhibited the upregulation of both protein and mRNA levels of p16, p21, and p53 in PQ-induced senescent endothelial cells (Figure 2C; Figure S5I). Similarly, silencing TET3, but not TET2, inhibited the PQ-induced enhancement in p-γH2AX intensity within endothelial cells (Figure 2D). Moreover, overexpression of TET3, in contrast to TET2, resulted in elevated levels of p16, p21, and p53 in HUVECs (Figure 2E,F). This overexpression of TET3 also led to an increased intensity of p-γH2AX in endothelial cells (Figure 2G) and significantly reduced proliferation of endothelial cells (Figure 2H).

To substantiate the role of TET3 in cellular senescence, the CRISPR/Cas9 technique was employed to delete the TET3 gene from HUVECs. The deletion of TET3 markedly reduced the PQ-induced mRNA and protein levels of p16, p21, and p53 in HUVECs (Figure 3A,B). Additionally, TET3 deletion resulted in a decreased number of senescent HUVECs, as indicated by reduced β-galactosidase staining (Figure 3C), and promoted cell proliferation in HUVECs (Figure 3D). TET3 deletion also significantly reduced the PQ-induced mRNA levels of inflammatory cytokines (Figure 3E,F). Furthermore, the deletion of TET3 markedly suppressed the PQ-induced elevation in the levels of p-γH2AX and 5-hmC in endothelial cells (Figure 3G). These findings indicate a potential role for TET3 in cellular senescence.

Notably, TET3 expression was significantly upregulated in the heart, aorta, and kidney of mice treated with PQ (Figure 4A-B, and Figure S6A–C). Given that homozygosity for TET3 results in neonatal sublethality [36], to further investigate the influence of TET3 on PQ-induced aging mice, heterozygous TET3 mice were generated (Figure S6D). The results revealed that TET3 deficiency led to the inhibition of p21 and p53 in the heart and aorta of PQ-induced aging mice (Figure 4C,D; Figure S6E). Moreover, TET3 deficiency led to a reduction in β-galactosidase levels in the heart and aorta of PQ-induced aging mice (Figure S6F,G), as well as decreased levels of p-γH2AX and 5-hmC in the aorta, heart, and kidney following PQ treatment (Figure 4E,F; Figure S6H). These results reinforce the role of TET3 as a key driver of the cardiovascular senescence.

2.3 TET3 Upregulates p53 Expression Through the 5-hmC-Mediated SP1-ETS-1 Axis

To elucidate the potential mechanism of TET3 in cardiovascular senescence, hMeDIP-seq was conducted in HUVECs with TET3 deletion. The analysis indicated that the levels of 5-hmC were predominantly localized at the transcriptional start site (Figure S7A). Importantly, despite the deletion of TET3, the distribution of 5-hmC across various genomic regions, including intergenic, intronic, upstream, promoter, and exonic regions, remained largely unchanged (Figure S7B). Subsequently, the focus was directed toward differentially hydroxymethylated sites (DhMSs) that were upregulated in PQ-induced HUVECs and downregulated following TET3 deletion. A total of 13,667 upregulated DhMSs were identified in PQ-induced HUVECs, while 5147 downregulated DhMSs were identified in HUVECs with TET3 deletion, each exhibiting a fold change of >2, p < 0.005, and false discovery rate < 0.05 (Figure 5A; Figure S7C, Table S3). Furthermore, the proportion of DhMSs across different genomic regions (intergenic, intron, upstream, promoter, and exon) remained nearly consistent despite the deletion of TET3 (Figure 5B).

The GO and Kyoto Encyclopedia of Genes and Genome (KEGG) analyses revealed the involvement of different hydroxymethylated genes (DhMGs) in the regulation of aging, inflammation, and cancer. Specifically, PQ stimulation enhanced various signaling pathways related to viral infections, inflammatory responses, and multiple cancers (Figure S7D-E). Knockout of TET3 inhibited the renin–angiotensin pathway, aldosterone synthesis and secretion, mitochondrial autophagy, and longevity-regulating pathway. It downregulated the inflammatory response to leukocyte migration, RAS signaling transduction, and telomere DNA-binding (Figure S7F,G). Furthermore, 5-hmC may regulate gene expression by facilitating modifications of promoter CpG sites [37, 38]. In our study, we conducted an analysis of DhMGs in gene promoters, selecting those with a fold change greater than 2, a p-value less than 0.005, and a false discovery rate below 0.05. Using a Venn diagram, we identified two overlapping DhMGs associated with senescence, namely SP1 and lysine acetyltransferase 5 (KAT5) (Figure 5C; Table S4). Although the levels of both SP1 and KAT5 were significantly increased in PQ-induced senescent cells (Figure S8A,B), TET3 knockdown only significantly inhibited SP1 expression, not KAT5 (Figure 5D,E). Moreover, the 5-hmC level of the SP1 promoter was significantly increased, and TET3 knockout significantly reduced its level (Figure 5F). These results indicated that TET3 may regulate SP1 expression in a 5-hmC-dependent manner.

Previous studies have demonstrated that SP1 can regulate transcription through cooperative interactions with ETS-1 [39-43], which has been associated with human aging and the levels of senescence-associated proteins [44, 45]. Therefore, the level and function of ETS-1 were verified in cardiovascular senescence, and results revealed that mRNA and protein levels of ETS-1 were significantly increased in the replicative senescent and the PQ-induced senescent HUVECs (Figure S8C–F). The combination of SP1 and ETS-1 was significantly increased in PQ-induced senescent cells, whereas TET3 knockdown significantly reduced this combination and the level of ETS-1 (Figure 5G). Moreover, ETS-1 knockdown significantly reduced the levels of p16, p21, and p53 in PQ-induced senescent cells (Figure 5H,I).

ETS-1 is known to induce cellular senescence by upregulating p53 expression [46, 47]. In this study, both TET3 knockdown and deletion significantly inhibited p53 expression (Figures 2, 3), whereas TET3 overexpression led to a marked increase in p53 expression (Figure 2E,F). These observations led us to hypothesize that TET3 modulates p53 expression via its interaction with the SP1-ETS-1 axis. To evaluate this hypothesis, we conducted a ChIP assay, which demonstrated a significant increase in SP1 enrichment at the p53 promoter in PQ-induced senescent cells (Figure 6A). Additionally, the enrichment of ETS-1 at the p53 promoter was also significantly increased in the replicative senescent and the PQ-induced senescent HUVECs (Figure 6B,C). TET3 knockdown notably diminished the enrichment of SP1 and ETS-1 at the p53 promoter (Figure 6D,E). Additionally, our findings indicated that TET3 deficiency reduced the levels of SP1 and p53 in the heart and aorta of aging mice induced by PQ (Figure 6F,G). These results suggested that TET3 regulated the expression of p53 by mediating the SP1-ETS-1 axis.

2.4 Interaction Between p53 and TET3 in Controlling Cellular Senescence

The role of p53 in senescence and aging has been well established [48]. In the present study, the knockdown of p53 resulted in decreased levels of p16 and p21 (Figure 7A,B). To further elucidate the impact of p53 on aging in vivo, p53-heterozygous mice were generated (Figure S9A). Our findings demonstrated that mRNA levels of p21 were significantly reduced in the hearts, kidneys, and aortas of p53^+/−-heterozygous mice induced by PQ treatment (Figure 7C,D; Figure S9B,C). Moreover, the protein levels of p16, p21, and β-galactosidase were also markedly reduced in the hearts and aortas of p53^+/−-heterozygous mice (Figure 7E–G).

TET3 affected the expression of p53 through the 5-hmC-mediated SP1-ETS-1 axis. Further analysis revealed a binding element of p53 at the proximal end of the TET3 promoter. To investigate whether p53 can also negatively regulate TET3 expression [49], a ChIP assay was performed, revealing that p53 binds to the TET3 promoter (Figure 8A). Furthermore, RNA polymerase II was also observed to bind to the p53 binding site on the TET3 promoter (Figure 8B). In addition, the results of the luciferase reporter assay for the TET3 promoter revealed that p53 overexpression or silencing significantly activated or prevented TET3 promoter activity (Figure 8C,D; Figure S10A,B). P53 knockdown also significantly reduced the levels of TET3 in PQ-induced senescent cells (Figure 8E; Figure S10C). The levels of TET3 were decreased in the hearts, kidneys, and aortas of p53^+/−-heterozygous mice induced by PQ (Figure 8F–H; Figure S10D–E). Furthermore, the levels of p-γH2AX and 5-hmC were significantly decreased in the aortas, hearts, and kidneys of p53^+/−-heterozygous mice that received PQ treatment (Figure 8I,J; Figure S9D). These data revealed the interdependence of p53 and TET3 in controlling cardiovascular senescence.

3.1 Summary

This study demonstrated that TET3 played a critical role in regulating cardiovascular senescence. These findings offer the new perspective that TET3 plays a role in cardiovascular senescence. Targeting TET3 activity is a potential therapeutic strategy for age-related cardiovascular diseases.

4.1 Experimental Animal Model

C57BL/6J male mice were purchased from Shanghai SIPPR-BK Laboratory Animal Co. Ltd (Shanghai, China). TET3-heterozygous and p53-heterozygous mice were commercially obtained from the Shanghai Model Organisms Center, Inc. (Shanghai, China). Because the TET3 homozygote may lead to neonatal sublethality, we used TET3-heterozygous mice for the experiments.

The mice were housed in a specific pathogen-free animal facility. Six-week-old male mice, including C57BL/6J, TET3^+/− mice, and p53^+/− mice, were intraperitoneally injected with PQ (Yuanye Biotechnology, Shanghai) at a dose of 10 mg/kg per week or phosphate-buffered saline (control). All the mice were euthanized after 4 weeks. Subsequently, the hearts, kidneys, and aortas were excised, weighed, and cryopreserved with liquid nitrogen, with a portion of the tissues sectioned for immunofluorescence.

4.2 Cell Line and Culture

HUVECs (CRL-1730) and human vascular smooth muscle cells (T/G HA, CRL-1999) were purchased from the American Type Culture Collection. HEK 293T cells, human kidney-2 (HK-2), and rat H9C2 cardiomyocytes were obtained from the Cell Bank of the Chinese Academy of Sciences.

HUVECs were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% newborn calf serum. The H9C2 cardiomyocytes and HEK293T cells were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. T/G HA cells were cultured in F-12K medium and HK-2 cells were cultured in K-SFM medium according to the manufacturer's protocol. The cell lines were cultured in a humid environment with 5% CO₂ and 95% air at a temperature of 37°C.

4.3 Senescent Cell Model

Replicative senescence in HUVECs was induced by subculturing the cells to a population doubling level (PDL) of 50 through repeated divisions [3]. Young cells were subcultured to a PDL of 10. PDL refers to the total number of times the cells in the population have doubled since their primary isolation in vitro. The PDL was calculated as follows: PDL = 3.32 (log UCY − log l) + X, where n is the final PDL at the end of a given subculture, UCY is the cell yield at that point, l refers to the cell number used as inoculum to begin that subculture, and X denotes the doubling level of the inoculum used to initiate the quantitated subculture. The cumulative population doubling was calculated as the sum of all the changes in population doubling during the subculturing process. Subsequently, the HUVECs were harvested for senescent staining, immunofluorescence, immunoblot, and quantitative polymerase chain reaction (qPCR) analyses.

To establish a PQ-induced senescent cell model, HUVECs were plated on 6-well plates with a density of 5 × 10⁵ cells per well, and cultured with PQ (20 µM) for 2, 3, and 4 days. Cells were cultured in the complete medium and served as negative controls. The cell proliferation was conducted following the manufacturer's instructions, utilizing the cell counting kit-8 (Beyotime Biotech, Nantong, China). Additionally, the staining of β-galactosidase was performed to identify cellular senescence and the expression of senescence-related genes was also measured. Moreover, the induction of cellular senescence by 20 µM PQ was tested in HK-2, T/G HA, H9c2, and HEK293T cells after a prolonged incubation duration of 3 days. Furthermore, gene-modified HUVECs of p53 or TET overexpression, knockout, and knockdown were treated with 20 µM PQ for 3 days.

4.4 MeDIP-seq and hMeDIP-seq

To investigate the specific genomic regions where the levels of 5mC and 5-hmC were altered in senescent endothelial cells, MeDIP-seq and hMeDIP-seq were performed. The MeDIP-seq and hMeDIP-seq process included labeling, hybridization, scanning, normalization, and data analysis, which was conducted by KangChen Biotech Inc. (Shanghai, China) and CloudSeq Biotech Inc. (Shanghai, China).

4.5 TET3 knockout by Targeting the CRISPR/cas9 Gene

For TET3 knockout (KO) in HUVECs, the TET3 CRISPR/Cas9 KO plasmid(h) (sc-414997) and TET3 HDR plasmid(h) (sc-414997-HDR) were used, with the control CRISPR/Cas9 plasmid (sc-418922) as negative control, all of which were purchased from Santa Cruz. The HUVECs were seeded in a 24-well plate at a density of 2 × 10⁵. Transfection of these plasmids was performed using FuGENE HD transfection reagent (Promega), and the cells were incubated for 6 h. After transfection, the cells were cultured in a fresh normal medium and subjected to puromycin selection (1 µg/mL) to enrich positive cells according to previous studies [68, 69]. After puromycin selection, the survival cells were plated on 96-well plates at a single-well density, and cell clones were screened using qPCR to confirm successful TET3 gene knockout.

4.6 shRNA Transfection

For gene silencing, 8 µg of shRNA plasmids (h) targeting TET2 (sc-88934-SH), TET3 (sc-94636-SH), p53 (sc-29435-SH), and ETS-1 (sc-29309) were individually transfected into HUVECs. Additionally, a shRNA plasmid-control (sc-108060) was used as a control for comparison. After 6 h of transfection, 20 µM PQ was added to the cell culture medium. Subsequently, after 3 days, the cells were harvested for functional experiments.

4.7 Transfection of Gene Overexpression

To overexpress human TET2, TET3, or p53 genes, their cDNA was inserted into the pCDH-CMV-MCS-EF1-Green plasmid (CD513B-1). The cells were placed in a six-well dish with a concentration of 5 × 10⁵ cells per well. Next, the cells were transfected with 8 µg of the TET2, TET3, or p53 plasmid or control plasmid by using the FuGENE HD transfection reagent (Promega) following the protocol established in previous studies [68, 69]. After 6 h of transfection, the transfection medium was replaced with a fresh normal medium, and the cells were incubated in a normal medium for an additional 24 h before protein or RNA extraction.

4.8 β-Galactosidase Senescence Staining

The cellular senescence assay kit (Cell Biolabs, Inc.) was used to stain senescent cells after fixing them in 4% paraformaldehyde. Staining was allowed to occur overnight at 37°C. After staining, the staining solution was removed. After incubation for 24 h, photographs of the stained cells were taken using a standard light microscope (Olympus).

4.9 RNA Extraction and qPCR

Total RNA of snap-frozen tissues or cultured cells was extracted by using TRIzol reagent (Thermo Fisher Scientific). The cDNA reverse transcription kit (Thermo Fisher Scientific) was utilized for cDNA synthesis as per the manufacturer's guidelines. QPCR analyses were performed. All primers (Table S5) used were obtained from Generay (Generay Biotech, Shanghai). Values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative expression values were calculated using the 2^−ΔΔCt method, and the results are expressed as relative fold changes.

4.10 Western Blot Analysis

To perform western blot analysis, snap-frozen tissues and cells were both homogenized in radioimmunoprecipitation assay lysis buffer (Beyotime Biotechnology, Nantong, China). After centrifugation, the lysates were quantified to prepare for gel electrophoresis. Samples were loaded and separated and then transferred onto NC membranes (HATF04700, Merck Millipore). Next, the membranes were blocked for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. Primary antibodies against p16 (1:1000, ab108349, Abcam), p21 (1:250, sc-397, Santa Cruz), p53 (1:1000, 2524, CST), TET2 (1:250, sc-136926, Santa Cruz), TET3 (1:250, sc-139186, Santa Cruz), SP1 (1:1000, 9389, CST), ETS-1 (1:1000, 14069, CST), STAT3 (1:1000, 9139, CST), p-STAT3 (1:1000, 9145, CST), β-actin (1:5000, 1702–67, Huabio), and GAPDH (1:5000, KC-5G5, KangChen Bio-tech Inc., Shanghai, China) were used. Proteins were detected and quantified using ImageJ software.

4.11 Dot Blot

The extraction of genomic DNA was performed using the genomic DNA mini preparation kit (D0063, Beyotime Biotech). The DNA samples were subjected to denaturation at a temperature of 99°C for 5 min. Subsequently, the denatured samples were applied onto nitrocellulose membranes. The membranes were treated using ultraviolet for 30 min and blocked with 5% nonfat milk for 2 h, and incubated with 5-hmC antibodies (1:500, ab106918, Abcam) overnight at 4°C. Then, HRP-conjugated goat antimouse antibodies were applied and then captured. To track DNA loading, membranes were stained using a solution of 0.02% methylene blue in 0.3 M sodium acetate (with a pH of 5.2).

4.12 Immunofluorescence Staining

The heart, kidney, and aorta tissues were fixed with 4% paraformaldehyde overnight. The tissues were then dehydrated and embedded in paraffin for sectioning. Before staining, the samples were treated with 4% paraformaldehyde for 15 min, followed by permeabilization with 0.5% triton X-100 for 15 min. Then the tissue sections or cells were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 h. The primary antibodies used were against 5-hmC antibody (1:200, 39769, Active Motif), p-γH2AX antibody (1:200, ab26350, Abcam), SP1 (1:100, A19649, ABclonal), TET3 (1:200, GTX-121453, GENETEX), β-galactosidase (1:100, 27198, CST), p16 (1:100, sc-1661, Santa Cruz), p21 (1:100, sc-6246, Santa Cruz), and p53 (1:100, 2524, CST). The stained cells were observed and imaged using an Olympus IX71 fluorescence microscope (Olympus). Images were processed using the cellSens software (Olympus) and quantified using ImageJ software.

4.13 Luciferase Reporter Assay

HEK 293T cells were transfected with p53 shRNA plasmid (sc-29435-SH, Santa Cruz) or human p53 overexpression plasmid for 24 h. Then, the 293T cells were cotransfected with a mixture of the TET3 promoter plasmid and the pRL-SV40 plasmid containing renilla luciferase gene. After 6 h of cotransfection, the culture medium was replaced, and the cells were cultured for 16 h. The luciferase assays were conducted utilizing the dual-luciferase reporter assay kit (E1980, Promega) following previous studies [69, 70], and the luciferase activity was measured using a Varioskan Flash microplate spectrophotometer (Thermo Scientific).

4.14 Immunoprecipitation Assay

The immunoprecipitation assay was performed using an assay kit (YJ003, EpiZyme, China) according to a previous study [71]. Briefly, the cells were prepared and homogenized for 10 min. The lysates were then collected through centrifugation to remove cell debris. The samples were incubated with protein A/G magnetic beads and SP1 (9389, CST) antibody overnight at 4°C. Finally, the samples were washed and denatured for subsequent western blotting analysis.

4.15 Chromatin Immunoprecipitation Assay

The ChIP assay was performed using an assay kit (ab500, Abcam). Briefly, the cells were crosslinked with 1% formaldehyde for 10 min. 3 × 10⁶ cells were lysed for 10 min. The chromatin was sheared through sonication performed ten times for 40 s, and then immunoprecipitated with the 2 g of antibodies targeting p53 (2524, CST), RNA polymerase II (ab300575, Abcam), SP1 (9389, CST), ETS-1 (14069, CST) and STAT3 (9139, CST) overnight at 4°C. The crosslinking was reversed at 65°C overnight, and the samples were digested with 100 µL of proteinase K at 45°C for 1 h. DNA was then recovered for qPCR or semiquantitative PCR. The semiquantitative PCR products were analyzed through electrophoresis on a 1.5% agarose gel.

4.16 Statistical Analysis

Statistical analysis was performed using GraphPad Prism version 9.0. Differences among the groups were compared using a one-way analysis of variance followed by Dunnett's multiple comparisons test or two-tailed Student's t-test. A p-value of <0.05 was considered statistically significant.