Clinical specimens and tissue microarray analysis

To investigate CLIP170 expression in RCC, we obtained cancerous and corresponding paracancerous tissues from 10 RCC patients at the Department of Urology, Second Affiliated Hospital of Dalian Medical University, for subsequent immunohistochemical analysis. Additionally, we created a tissue microarray (TMA) using specimens from 145 RCC postoperative cases (Table S1), collaboratively produced in partnership with Shanghai Outdo Biotech Company (Outdo Biotech, Shanghai, China). Ethical approval for this study's protocol was granted by the ethics committee of the Second Affiliated Hospital of Dalian Medical University (Approval No. DYEY-2023-184), and all participating patients provided informed consent.

Immunohistochemistry (IHC) analysis

IHC was performed as previously described [11]. Briefly, tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Endogenous peroxidase activity was blocked using 3% hydrogen peroxide solution at room temperature for 15 min. After blocking with 10% goat serum at 37 °C for 30 min, sections were incubated overnight with primary antibodies at 4 °C. The list of antibodies used is summarized in Table S1. Staining intensity in cancer cells was evaluated as follows: score 0 = negative staining intensity, 0.5 = very weak staining intensity, score 1 = weak staining intensity, score 2 = moderate staining intensity, and score 3 = strong staining intensity. The proportion of positive areas ranged from 0 to 100%. The immunohistochemical staining score was determined by multiplying the staining intensity score by the proportion of the positive area (0–300%). The stained slides were independently evaluated and scored by two experienced pathologists who reached a consensus.

Cell culture

Four human renal cancer cell lines (786-O, CAKI-1, ACHN, and 769-P) and a human normal proximal tubular epithelial cell line (HK-2) from Procell company (Procell, Wuhan, China) were obtained (passage number of cell lines: 5–20) and authenticated via short tandem repeat identification. These cell lines were cultured in a humidified atmosphere with 5% CO2 at 37 °C and were maintained in appropriate media: RPMI-1640 medium for 786-O and 769-P cells, 5A medium for CAKI-1 cells, and MEM for ACHN cells. The culture media were supplemented with 10% fetal bovine serum (BI, Kibbutz, Israel) and 1% penicillin/streptomycin (Invitrogen, CA, USA).

RNA interference and cell transfection

Small interfering RNAs designed according to the gene sequence were synthesized by GenePharma Co., Ltd. (GenePharma, Suzhou, China). The diluted RNAs were combined with Lipofectamine 3000 transfection reagent (Invitrogen, CA, USA) following the manufacturer's instructions before being introduced into the target cells for transfection. Lentiviruses carrying short hairpin RNAs were generated by Genechem Co., Ltd. (Genechem, Shanghai, China). Following lentivirus infection for 48 h, 2 μg/mL puromycin (Beyotime, Shanghai, China) was added to the cell culture medium for 2 weeks to select stable cell lines. The target sequences are listed in Table S2.

RNA extraction and reverse transcription quantitative real-time PCR (RT-qPCR)

Cell and tissue RNA extraction was performed using TRIzol reagent (Invitrogen, CA, USA). Subsequently, reverse transcription was carried out following the prescribed protocol, utilizing the MonScript RTIII All-in-One Mix to generate complementary DNA (Monad Biotech, Wuhan, China). To quantify gene expression levels, RT-qPCR was conducted using the MonAmp™ ChemoHS Specificity Plus qPCR Mix (Monad Biotech, Wuhan, China) with the Bio-Rad CFX96 Real-Time PCR detection system (Bio-Rad, CA, USA). Specific primer pairs were designed and synthesized by Sangon Biotech Company (Sangon Biotech, Shanghai, China) and are listed in Table S3.

Western blot

Total protein purification was accomplished using lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitors (Beyotime, Shanghai, China). The protein concentration was determined using the bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). Protein samples were separated using SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred electrophoretically onto a PVDF membrane. After blocking for 2 h with 5% non-fat dry skim milk, the membrane was incubated overnight at 4 °C with the corresponding primary antibodies (Table S1), followed by treatment with secondary antibodies, including anti-mouse immunoglobulin G (IgG; 1:5000, SA00001-1, Proteintech, Wuhan, China) and anti-rabbit IgG (1:5000, SA00001-2, Proteintech, Wuhan, China) at room temperature for 2 h. Chemiluminescence was detected using Tanon™ High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China) and visualized using the Clinx ChemiCapture system (Clinx Science Instruments, Shanghai, China).

CCK-8 cell viability assay

A total of 3,000 cells were seeded in each well of 96-well plates. After incubation at specified time points (0, 24, 48, and 72 h), a tenfold diluted cell counting Kit-8 reagent (APExBIO, Houston, USA) was added and incubated for 2 h at 37 °C. Subsequently, the absorbance at 450 nm was measured using a multifunctional microplate reader (Tecan, Männedorf, Switzerland).

Colony formation assay

In 12-well plates, 250 cells were cultured for a period of 2 weeks. Afterward, the cells were fixed with a 4% paraformaldehyde solution for 30 min and then stained with a 0.1% crystal violet solution (Coolibo, Beijing, China) for an additional 30 min. Following three washes with phosphate-buffered saline (PBS), colony images were captured and documented, along with the corresponding colony counts.

5'-Ethynl-2'-deoxyuridine (EdU) assay

RCC cells were seeded onto 12-well plates equipped with cell climbing slides. To assess cell proliferation, an EdU staining assay was conducted using the BeyoClick™ EdU-488 Cell Proliferation Assay Kit (Beyotime, Shanghai, China), following the manufacturer's protocol. In brief, RCC cells were incubated in a 10 μM EdU solution for 2 h, subsequently fixed with 4% paraformaldehyde, and permeabilized using PBS containing 0.3% Triton X-100. Following this, the cell climbing slides were shielded from light and incubated with the EdU reaction mixture at 37 °C for 30 min. Cell nuclei were counterstained with 4ʹ,6-diamidino-2-phenylindole (DAPI). Finally, cells were visualized using fluorescence microscopy (Leica, Nussloch, Germany), and the proportion of EdU-positive cells was calculated as the number of EdU-positive cells (green) divided by the number of DAPI-positive cells (blue).

Cell migration and invasion assays

To assess cell migration, wound-healing assays were employed. RCC cells were cultured in 12-well plates until reaching approximately 85% confluence, at which point a linear wound was generated by gently scraping the cell surface with the tip of a 200 μL pipette. Cell migration was monitored at 0 and 24 h post-scraping. For the evaluation of cell migration and invasion capabilities, Transwell chambers (Corning, NY, USA) were utilized. RCC cells were seeded into the upper chambers in FBS-free medium, with or without a Matrigel coating diluted fivefold in FBS-free medium. The lower chambers were filled with medium containing 20% FBS. After 36 h of incubation, the chambers were removed, and the cells were fixed with 4% paraformaldehyde for 30 min. Subsequently, cells were stained with 0.1% crystal violet (Coolibo, Beijing, China) for 15 min. Three visual fields were randomly selected for counting migrated or invasive cells.

4D-label free proteomics analysis

After transfection, cells were thoroughly lysed using a protein lysis buffer (8 M urea, 100 mM triethylammonium bicarbonate buffer, pH 8.5) followed by sonication in an ice bath. The lysates were centrifuged, and the supernatant was collected. Proteins were reduced using 1 M dithiothreitol at 56 °C for 1 h and subsequently alkylated with iodoacetamide at room temperature for 1 h in the dark. The reaction was quenched by cooling the samples on ice. Protein concentrations were measured using the Bradford Protein Assay Kit (Beyotime, Shanghai, China) to ensure equal loading across all samples.

For each sample, SDS-PAGE gel electrophoresis was performed, followed by Coomassie brilliant blue staining to assess protein integrity. The protein samples were then digested with trypsin in the presence of calcium chloride. After digestion, the peptides were desalted and dried by lyophilization.

For liquid chromatography, mobile phase A (100% water with 0.1% formic acid) and mobile phase B (100% acetonitrile with 0.1% formic acid) were prepared. The lyophilized peptides were reconstituted in mobile phase A and separated using a Waters BEH C18 (4.6 × 250 mm, 5 μm) column in an L-3000 HPLC system (Scilogex, Rocky Hill, USA) at 45 °C. Peptide fractions were collected at 1-min intervals, combined into 10 fractions, and lyophilized.

Subsequently, ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS) was performed using a nanoElute UHPLC system (Bruker, Bremen, Germany) coupled with a timsTOF pro2 mass spectrometer (Bruker, Bremen, Germany). Two hundred nanograms of peptides, dissolved in 0.1% formic acid, were injected for mass spectrometric analysis. MS data were acquired in data-dependent acquisition (DDA) mode, with a dynamic exclusion time set to ensure comprehensive proteome coverage.

Following mass spectrometry data acquisition, spectral data were processed using the Proteome Discoverer (Thermo Fisher, CA, USA) and MaxQuant (Bruker, Bremen, Germany) search engines, with peptide identification based on the UniProt human database. A false discovery rate (FDR) of < 1% was applied at both the protein and peptide levels. Protein quantification was conducted using the Label-Free Quantification (LFQ) method implemented in MaxQuant.

Bioinformatics data analysis

To ensure data accuracy and reduce technical variability, raw proteomics data were further processed in R 3.6.3 software. First, LFQ intensities were log2-transformed for normalization. Proteins with more than 50% missing values across samples were filtered out, and any remaining missing values were imputed using the missForest algorithm. This imputation method uses a machine learning-based approach to improve the robustness of the dataset. To identify differentially expressed proteins between the experimental and control groups, we employed the limma package in R. Proteins with an adjusted P-value (FDR < 0.05) and a log2 fold change (|log2FC|> 2) were considered significantly different. These criteria ensured the identification of proteins with biologically meaningful differences in expression.

And then, several visualization techniques were used to enhance data interpretation. A heatmap of the differentially expressed proteins was generated using the pheatmap package, applying hierarchical clustering to both proteins and samples, with color intensity reflecting protein expression levels. A volcano plot, generated with the EnhancedVolcano package, visually displayed the relationship between fold change and statistical significance, highlighting significantly different proteins based on FDR and fold change. To investigate the signaling pathways regulated by CLIP170, Gene Set Enrichment Analysis (GSEA) was performed using the clusterProfiler package in R. A ranked list of proteins based on log2 fold change was used for the analysis. The GSEA was conducted against the KEGG databases, with significance determined by an FDR threshold of < 0.05.

Co-immunoprecipitation (Co-IP) assay

Five hundred milligrams of extracted total protein were gently agitated and incubated in the presence of 5 µg of primary antibodies or IgG for 2 h at room temperature. Following this incubation, 15 µL of BeyoMag™ Protein A + G magnetic beads (Beyotime, Shanghai, China) were added and allowed to incubate at 4 °C overnight. Subsequently, the immunoprecipitates were subjected to elution and analyzed via western blotting.

Immunofluorescence (IF) assay

RCC cell climbing slices were fixed in 100% methanol for 15 min, followed by permeabilization with a 0.1% Triton X-100 solution. After blocking with 10% normal goat serum, primary antibodies for CLIP170 and β-catenin were incubated with the RCC cells at 4 °C overnight. The following day, fluorescent secondary antibodies, Alexa Fluor 647 anti-mouse (1:500, A0473, Beyotime, Shanghai, China), and Alexa Fluor 488 anti-rabbit (1:500, A0423, Beyotime, Shanghai, China), were used to incubate the slices for 2 h each. After DAPI staining, a fluorescence microscope was used for visualization.

Protein–protein docking

To predict the direct binding model between CLIP170 (UniProt accession number: P30622) and β-catenin (UniProt Accession Number: P35222) human origin protein molecules, we initiated the process by retrieving their respective crystal structure using the UniProt database (https://www.uniprot.org/). The full-length crystal structure of the CLIP170 protein has not yet been resolved; therefore, the three-dimensional structure predicted by AlphaFold2 was utilized. For the β-catenin protein, the A chain from the 1G3J crystal structure, sourced from the PDB database (https://www.rcsb.org/), with a resolution of 2.1 Å, was employed. Subsequently, various operations were performed on both CLIP170 and β-catenin proteins, including bond assignment, hydrogenation, zero-level bond assignment to metal atoms, and creation of disulfide bonds. Following these steps, hydrogen bond networks were optimized, and finally, protein energy minimization was conducted using the OPLS_4 force field. Then, molecular docking of protein–protein interactions was performed using the protein–protein docking module (Piper) within the Schrödinger tool (https://www.schrodinger.com/). Conformational mapping and docking region analysis were performed using PYMOL (https://pymol.org/) for comprehensive investigation and visualization of the binding interactions.

Xenograft tumor assay

Four-week-old female BALB/c nude mice were obtained from the Weitong-Lihua Experimental Animal Center (Weitong Lihua, Beijing, China) and housed in a specific pathogen-free environment. Xenograft tumor mouse models were established by subcutaneously injecting 2 × 106 RCC cells into the mice's right-behind armpit to evaluate subcutaneous tumor growth in the Vector, CLIP170, shRNA-V, and shCLIP170 groups. Tumor volume was assessed through periodic measurements of tumor length and width at 5-d intervals using Vernier calipers. Tumor volume was calculated as length × width^2/2, with a maximum limit of 1000 mm3 for tumor burden. After a 30-d period, the mice were humanely euthanized, and their tumors were extracted, weighed, and fixed in 4% paraformaldehyde for 48 h. The tumor specimens were then paraffin-embedded and sectioned for subsequent histochemical staining. All animal-related protocols received prior approval and were conducted in strict accordance with the stipulations of the Animal Experimental Ethical Committee at the Laboratory Animal Center of Dalian Medical University (Approval No. AEE23056).