Fig 2: Verification of CDCP1 function in vitro. Transwell assays A and cell migration assays B showed that CDCP1 overexpression promoted the migration ability of glioma cells in vitro ( Wilcoxon rank-sum test)(the magnification is 200 times). Western blotting C was performed to detect the expression of N-cadherin, vimentin and Slug in the LV-CDCP1 and LV-Con groups

Fig 3: CDCP1 mediates epithelial-to-mesenchymal transition (EMT) in cervical cancer cells in vitro.Notes: (A and B) Western blot was used to confirm the expression of E-cadherin/vimentin protein after transfected with shRNA or pReceiver-M07 vector which indicates that CDCP1 increased EMT in CC cell lines. Results are representative of at least three independent experiments showing similar results. All the data above presented as mean ± SD, *P<0.05.Abbreviations: CDCP1, CUB domain-containing protein-1; SiHa, Caski, C33A, HeLa, cervical cancer cell lines; shRNA, shCDCP1, short hairpin RNA targeting CDCP1; NC, negative control; CC, cervical cancer; SD, standard deviation; EMT, epithelial-to-mesenchymal transition.

Fig 4: Identified CDCP1 correlated with poor prognosis and involved in regulating CTL function in cervical cancer. (A) Kaplan-Meier survival curves showing the overall survival of patients based on CTL scores (average expression of CD8A, CD8B, GZMA, GZMB, and PRF1) in TCGA-CESC(The Cancer Genome Atlas-Cervical squamous cell carcinoma and endocervical adenocarcinoma) Cohort. The log-rank test was used to determine the statistical significance of the difference between CDCP1 high/low expression groups, with p<0.05 considered statistically significant. (B) Volcano plot depicting the analysis of differentially expressed proteins between the CTL low-score group and the CTL high-score group. Each point corresponds to a gene, with genes exhibiting significant differential expression highlighted in red (upregulated in the CTL low-score group) and blue (downregulated in the CTL low-score group). Genes with |log2FC| greater than 2 and p<0.01 are considered significantly differentially expressed. Genes encoding membrane proteins that show significant differential expression are specifically labeled, including ITGAL, PTPRC, PTPRN, WAS, and CDCP1. (C) Schematic representation of systematic screening for immunotherapy targets in cervical cancer using the TCGA database. Through survival analysis and differential gene analysis based on immune scores from the TCGA cervical cancer dataset, membrane protein-coding genes associated with patient prognosis and low CTL scores were identified as potential targets for immunotherapy. (D) qRT-PCR analysis of CDCP1 mRNA expression levels in various patients sample.(E) Western blot analysis of NC (n=12) and CC (n=12) tissue samples. (F) Representative IHC images of CDCP1 staining in the NC and CC tissue samples. Bar in ×100, ×200, ×400 magnifications=200 µm, 100 µm, 50 µm, respectively. (G) Quantification of low and high CDCP1 expression percentages in the NC (n=30) and CC (n=176) groups based on the IHC staining results. Data are mean±SEM. Significance was determined using Student’s t-test, “ns”: not significant, **p<0.01, ****p<0.0001. CC, cervical cancer (n=56); CTL, cytotoxic T lymphocyte; IHC, immunohistochemistry; NC, normal cervix (n=35).

Fig 5: The expression level of CDCP1 effected xenograft tumor growth in an immune-dependent manner. (A, B) Subcutaneous injection of control cells, CDCP1 knockdown, and CDCP1 overexpression U14 cells into 5-week-old female BALB/c-nu immunodeficiency mice (n=8 for each group). Representative images are shown in A, B. The line graph depicts the tumor growth curve of mice. Scale bar:50 mm. Values are presented as the mean±SD. *p<0.05, **p<0.01. (C, D) Subcutaneous injection of control cells, CDCP1 knockdown, and CDCP1 overexpression U14 cells into 6-week-old female C57BL/6J immunocompetent mice (n=8 for each group). Representative images are shown in (C, D). The line graph shows the tumor growth curve of mice. Scale bar:50 mm. Values are presented as the mean±SD ns, not significant. *p<0.05, **p<0.01. (E–G) Proliferation of control cells, CDCP1 knockdown, and CDCP1 overexpression U14 cells analyzed by CCK-8 assay (E) and colony formation assay (F, G). Data represent at least three independent experiments. Values are presented as the mean±SEM. Significance was determined using one way ANOVA test, *p<0.05, **p<0.01. ANOVA, analysis of variance; ns, not significant.