The UALCAN (https://ualcan.path.uab.edu/index.html) and GEPIA (gepia.cancer-pku.cn) databases were used to evaluate the expression of EBP1 in the tissues of patients with KIRC and its association with tumor grade, stage and overall survival. In GEPIA, the ‘match TCGA normal and GTEx data’ setting was used.

Preliminary data were obtained demonstrating that EBP1 is expressed at high levels in 786-O and 769-P human KIRC cell lines. Specifically, the mining and analysis of relevant public databases revealed that the expression level of EBP1 in these two cell lines was significantly higher compared with that in most other renal cancer cell lines, a trend consistently validated across multiple bioinformatics platforms, including the Cancer Cell Line Encyclopedia database (https://sites.broadinstitute.org/ccle/), The Cancer Genome Atlas (TCGA; https://www.cancer.gov/tcga; TCGA-KIRC project) and Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/). The GSE15641 (18) dataset was downloaded from the GEO.

Therefore, these cell lines were chosen for analysis. The 786-O and 769-P cell lines were sourced from the Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. They were cultured at 37°C in an atmosphere consisting of 5% CO₂ and 95% air, using RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) enriched with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), along with 100 µg/ml streptomycin and 100 U/ml penicillin. The 786-O and 769-P cells were seeded into culture dishes, and the medium was supplemented with doxorubicin (3 µM; cat. no. E2516; Selleck Chemicals) and the p38MAPK inhibitor SB203580 (10 µM; cat. no. S1076; Selleck Chemicals). All cells were incubated at 37°C with 5% CO₂ in a humidified incubator for 48 h to ensure effective drug treatment.

In order to assess the effect of doxorubicin on the viability of the 786-O and 769-P cell lines, 200 µl complete growth medium was added to the cells in each well of a 96-well plate, and the cells were then incubated for 24 or 48 h with 3 µM doxorubicin. The CCK-8 assay kit from Shanghai Biyuntian Biotechnology Co., Ltd. China was then used to analyze the cells. In summary, each sample was treated with 10 µl CCK-8 solution and incubated at 37°C for 2 h. The optical density at 450 nm was then assessed using a microtiter plate reader, and cell survival curves were plotted.

A colony formation assay was performed to assess the ability of doxorubicin to inhibit 786-O and 769-P cell proliferation. The cells were inoculated into 6-well plates at a density of 3×10³ cells/well and the plates were incubated for 1 week. After aspirating the liquid from the 6-well plate, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and washed with PBS. Subsequently, the cells were stained with Giemsa (Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 20 min, followed by two washes with PBS. Colonies were defined as containing ≥50 cells and were quantified using ImageJ bundled with Java8 64-bit (National Institutes of Health).

The scratch test was performed to determine the effect of doxorubicin on the migration of 786-O and 769-P cells. The 769-P and 786-O cells were seeded in 6-well plates at a density of 1×10⁶ cells/well and grown to 90% confluence. Subsequently, the cells were cultured overnight in serum-free medium. In brief, a sanitized grid scale was placed over the confluent cells in a 6-well plate, and a 1,000-µl pipette tip was applied from the top to bottom of the grid to create a scratch in the cells. The cells were washed twice with PBS and then cultured in serum-free medium containing doxorubicin for 48 h. Using a Nikon Eclipse Ti-S/L100 inverted phase contrast fluorescence Microscope (Nikon Corporation), images of cell migration into the wound were captured at 0, 24 and 48 h at the same lesion site. Wound measurement was performed using ImageJ, and statistical analysis was conducted using GraphPad Prism 10.0 (Dotmatics). Each experiment was repeated at least three times.

Apoptosis was assessed using the DeadEnd™ Fluorometric TUNEL System (Promega Corporation) and fluorescence microscopy. Six-well plates were seeded with 1×10⁵ 786-O or 769-P cells/well, and the cells were cultured at 37°C in an incubator with 5% CO₂. The cells were treated with doxorubicin for 24 h before analysis using the TUNEL assay kit. First, the cells were fixed with 4% paraformaldehyde at room temperature for 15 min, washed three times with PBS, then incubated with rTdT enzyme incubation buffer at 37°C for 70 min in the dark. Subsequently, the cells were stained with DAPI solution (1 ml/well) at room temperature for 15 min. The samples were then immediately examined under a fluorescence microscope and images were captured (magnification, ×20). Quantification was performed from ~10 fields of view using ImageJ, and statistical analysis was conducted using GraphPad Prism 10.0. Each experiment was repeated at least three times.

Cells were lysed and then washed twice with ice-cold PBS (pH 7.4). Protein lysates were extracted using RIPA buffer (Beijing Solarbio Science & Technology Co., Ltd.), and nuclear and cytoplasmic extracts were prepared using the Nuclear and Cytoplasmic Extraction Reagents Kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The total protein concentration of the lysate was determined using a BCA kit (Thermo Fisher Scientific, Inc.). The protein lysates (30 µg/lane) were then separated by SDS-PAGE on 8–10% gels to ensure effective protein separation and accurate quantification. The proteins were subsequently transferred to a PVDF membrane. The membranes were blocked using 5% skimmed milk (Yuanda Mountain Dairy Co.) in TBS with 10% Tween-20 (TBS-T) for 2 h at room temperature. After blocking, the membranes were incubated with the following primary antibodies in TBS-T overnight at 4°C: EBP1 (cat. no. sc-393114; Santa Cruz Biotechnology, Inc.), phosphorylated (p-)p38 (cat. no. sc-166182; Santa Cruz Biotechnology, Inc.), p38 (cat. no. sc-81621; Santa Cruz Biotechnology, Inc.), HIF-1α (cat. no. sc-53546; Santa Cruz Biotechnology, Inc.), Bcl-2 (cat. no. sc-7382; Santa Cruz Biotechnology, Inc.), Bax (cat. no. sc-70407; Santa Cruz Biotechnology, Inc.), cleaved-caspase 3 (cat. no. sc-373730; Santa Cruz Biotechnology, Inc.), caspase 3 (cat. no. sc-56053; Santa Cruz Biotechnology, Inc.) and β-actin (cat. no. sc-47778; Santa Cruz Biotechnology, Inc.). The primary antibodies were diluted 1:1,000 in antibody diluent buffer (Beyotime Institute of Biotechnology). Subsequently, the membranes were incubated with HRP-conjugated goat anti-mouse secondary antibodies (1:5,000; cat. no. A0216; Beyotime Institute of Biotechnology) at 4°C for 2 h. The proteins were visualized using the ECL Western Blotting Substrate system (Beijing Solarbio Science & Technology Co., Ltd.). Protein grayscale values were measured using ImageJ, and statistical analysis was performed using GraphPad Prism 10.0, with each experiment repeated ≥3 times.

The 786-O and 769-P cells were transduced with two different short hairpin RNA (shRNA) lentiviral vectors targeting EBP1 (sh-EBP1*1 and sh-EBP1*2) and two different shRNA lentiviral vector negative controls (sh-NC*1 and sh-NC*2; Shanghai GeneChem Co., Ltd.). The sh-EBP1 target sequence was 5′-ACCAGCATTTCGGTAAATA-3′, and the sh-EBP1 sequences were as follows: sh-EBP1*1 (PA2G4-RNAi(23071–1)-a), 5′-CCGGCCACCAGCATTTCGGTAAATACTCGAGTATTTACCGAAATGCTGGTGGTTTTTG-3′ and sh-EBP1*2 (PA2G4-RNAi(23071–1)-b), 5′-AATTCAAAAACCACCAGCATTTCGGTAAATACTCGAGTATTTACCGAAATGCTGGTGG-3′. The control gene insertion sequence was 5′-TTCTCCGAACGTGTCACGT-3′, and the sequences of the shRNA controls were as follows: sh-NC*1, 5′-CCGGTTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAATTTTTG-3′ and sh-NC*2, 5′-AATTCAAAAATTCTCCGAACGTGTCACGTCTCGAGACGTGACACGTTCGGAGAA-3′. An EBP1 overexpression lentiviral vector (pHS-AVC-LY124) and control vector (pHS-AVC-ZQ328) were purchased from SyngenTech Co., Ltd. Lentiviral infection was carried out immediately after virus preparation. The virus titers were as follows: shNC1, 8×10⁸ TU/ml; shNC2, 8×10⁸ TU/ml; sh1 (shEBP1*1), 7×10⁸ TU/ml; sh2 (shEBP1*2), 7×10⁸ TU/ml. The multiplicity of infection (MOI) values in 786-O cells for shNC1, shNC2, sh1 and sh2 were 5, 10, 10 and 15, respectively. In 769-P cells, the MOI values for shNC1, shNC2, sh1 and sh2 were 10, 15, 15 and 20, respectively. The lentiviruses were added to the cells and incubated for 24 h at 37°C for gene transduction. Prior to adding the virus to the cells, it was mixed with 2 µg/ml polybrene to enhance infection efficiency. Selection was performed with 1 g/ml puromycin to obtain successfully transduced cells. Transduction efficiency was assessed by western blotting immediately.

Images were analyzed using ImageJ software. Differences between two groups were compared by unpaired Student's t-test while differences among multiple groups were compared using one-way or two-way ANOVA with Tukey's post hoc analysis. Statistical evaluation of the results was performed using GraphPad Prism 10.0. Experimental results are presented as the mean ± SD. Each experiment was replicated ≥3 times. P<0.05 was considered to indicate a statistically significant difference.

The UALCAN and GEPIA databases were used to evaluate the expression of EBP1 in KIRC. The results showed a marked upregulation in EBP1 expression in KIRC tissues compared with normal tissues (Fig. 1A). The association between KIRC status and EBP1 expression was then assessed. The results revealed that EBP1 expression was highly associated with tumor grade (Fig. 1B). Moreover, the analysis of pathological staging results demonstrated that EBP1 expression was associated with the stage of KIRC (Fig. 1C). In addition, survival curves (Fig. 1D) showed that the survival rate of patients with high EBP1 expression was significantly lower compared with that of patients with low EBP1 expression, indicating a robust association between EBP1 expression and patient survival.

To clarify the regulatory role of EBP1 in KIRC, EBP1 in 786-O and 769-P cells was stably knocked down, and the transduction effect was verified using western blotting. The results revealed that sh-EBP1*1 had a stronger knockdown effect than sh-EBP*2. Therefore, sh-EBP1*1 and sh-NC*1 were selected for use in subsequent experiments (Fig. 1E).

CCK-8 assay results showed that 786-O and 769-P cells in the sh-EBP1 group grew more slowly than those in the sh-NC group, with a significant difference detected between the groups on days 4–6; following doxorubicin treatment, the viability of cells with EBP1 knockdown was significantly lower than that of the cells transduced with sh-NC (Fig. 2A). The scratch assay demonstrated that 786-O and 769-P cells in the sh-EBP1 group had a significantly lower capacity to repair scratches after receiving doxorubicin treatment for 24 and 48 h in comparison with those in the sh-NC group (Fig. 2B), indicating that the inhibitory effect of doxorubicin on the migratory ability of KIRC cells was stronger following the knockdown of EBP1. The results of the colony formation assay showed that the number of colonies formed by doxorubicin-treated 786-O and 769-P cells was lower in the sh-EBP1 group than in the sh-NC group (Fig. 2C). This result illustrates that the inhibition of KIRC cell proliferation by doxorubicin was enhanced following the knockdown of EBP1. TUNEL staining showed that few 786-O and 769-P cells underwent apoptosis in the sh-NC group treated with doxorubicin, and the proportion of apoptotic cells was significantly higher in the sh-EBP1 group (Fig. 2D). These results indicate that EBP1 knockdown promotes doxorubicin-induced cell death in KIRC cells, suggesting that the anticancer effects of doxorubicin may be potentiated by EBP1 knockdown.

The 786-O and 769-P cells were cultured in media containing doxorubicin (3 µM) and the p38MAPK inhibitor SB203580 (10 µM) at 37°C and 5% CO₂ for 48 h. When doxorubicin was administered to cells in the sh-NC group, the p-p38MAPK/p38MAPK ratio and HIF-1α expression levels significantly decreased (Fig. 3). In the sh-EBP1 group, significant reductions in the p-p38MAPK/p38MAPK ratio and HIF-1α expression levels were also observed after the addition of doxorubicin compared with those in the sh-NC group. In the simultaneous presence of doxorubicin and SB203580, the suppression of the p-p38MAPK/p38MAPK ratio and HIF-1α expression in the sh-NC group was greater compared with that achieved with doxorubicin alone; however, a stronger suppression was observed in the sh-EBP1 group. These results suggest that doxorubicin acts by inhibiting p38MAPK phosphorylation and HIF-1α expression.

The expression of apoptosis-associated proteins Bax and Bcl-2, and the cleavage of caspase-3 in doxorubicin-treated KIRC cells were examined using western blotting. When 786-O and 769-P cells were treated with doxorubicin, the proportion of caspase-3 that was cleaved and the expression of pro-apoptotic factor Bax increased significantly. Conversely, there was a notable downregulation in the expression of Bcl-2, an inhibitory apoptotic component. Furthermore, the Bax/Bcl-2 ratio increased significantly when SB203580 was combined with doxorubicin (Fig. 4A and B). These results suggest that the knockdown of EBP1 promotes doxorubicin-induced apoptosis in KIRC cells by altering the levels of apoptosis-associated proteins via the p38MAPK pathway.

The viability of KIRC cells in the sh-NC and sh-EBP1 groups following treatment with doxorubicin and SB203580 was assessed using the CCK-8 assay. When the sh-EBP1 group was compared with the sh-NC group in each cell line, it was observed that doxorubicin treatment reduced cell viability and that this effect was even more pronounced following the addition of SB203580 (Fig. 4C and D). These findings indicate that doxorubicin and EBP1 knockdown cooperate via the p38MAPK pathway to increase the inhibitory effect of doxorubicin on KIRC cell survival.