Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: PDRG1 is upregulated in HCC and associated with unfavorable prognosis. (A) Expression analysis of PDRG1 across multiple HCC datasets from the HCCDB database showing significant upregulation in tumor tissues compared with non-tumorous liver tissues. (B, C) Representative immunoblotting and IHC images of PDRG1 expression in paired HCC and adjacent noncancerous tissues from our cohort (n = 86). (D) The Cox regression analysis showed that in the public dataset, the higher the expression level of PDRG1, the worse the overall survival (OS), disease-free survival (DFS), disease specific survival (DSS), and progression-free survival (PFS). (E, F) OS and PFS analyses in our clinical cohort confirming that high PDRG1 expression predicts poor prognosis. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Expressing, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: PDRG1 regulates proliferation, migration, invasion, and EMT in HCC cells. (A) Western blot validation of PDRG1 knockdown efficiency in HuH-7 and HLF cells and overexpression in SNU449 cells. (B) Wound-healing assays showing impaired migration upon PDRG1 knockdown and enhanced migration upon PDRG1 overexpression. (C) Transwell migration and invasion assays demonstrating reduced motility and invasiveness after PDRG1 knockdown and increased motility after PDRG1 overexpression. (D) Colony formation assays showing decreased clonogenicity upon PDRG1 knockdown and increased colony-forming ability with PDRG1 overexpression. (E) EdU incorporation assays indicating decreased proliferation in PDRG1-silenced cells and elevated proliferation in PDRG1-overexpressing cells. (F) Western blot analysis of EMT markers showing increased epithelial markers (E-cadherin and β-catenin) and decreased mesenchymal markers (N-cadherin and Vimentin) after PDRG1 knockdown, with the opposite effect upon PDRG1 overexpression. All experiments were performed with three independent biological replicates (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Migration, Western Blot, Biomarker Discovery, Knockdown, Over Expression

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: Transcriptomic profiling reveals that PDRG1 suppresses tumor cell senescence and promotes HCC progression. (A) Volcano plot showing 696 differentially expressed genes upon PDRG1 overexpression. (B) Heatmap of the top significantly altered genes in PDRG1-overexpressing cells. (C) GSEA showing enrichment of senescence-related signatures in PDRG1-overexpressing cells. (D) Western blot analysis showing that PDRG1 knockout increases p21 and SASP factors (IL-6, IL-8, and IL-1β) while decreasing p-CDK2 and p-RB; conversely, PDRG1 overexpression reduces p21 and SASP factors and increases p-CDK2 and p-RB. (E) Xenograft assays show that SNU449 cells overexpressing PDRG1 display enhanced tumor growth, as evidenced by increased tumor volume and tumor weight. (F-H) IHC analysis of Ki-67, p-RB, p21, IL-6, and IL-1β in xenograft tumors showing reduced senescence-associated markers in PDRG1-overexpressing tumors. (I) CCK-8 assays showing that p21 restoration significantly attenuates proliferation in PDRG1-overexpressing SNU449 cells. (J) EdU incorporation assays showing that p21 restoration reduces DNA synthesis in PDRG1-overexpressing SNU449 cells. (K) Wound-healing assays showing that p21 overexpression reverses the enhanced migratory capacity induced by PDRG1 overexpression. (L) Transwell migration and invasion assays showing that p21 restoration suppresses PDRG1-induced increases in migration and invasion. (M) Colony formation assays showing that p21 overexpression markedly reduces clonogenic growth in PDRG1-overexpressing SNU449 cells. All experiments were performed with three independent biological replicates (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Over Expression, Western Blot, Knock-Out, CCK-8 Assay, DNA Synthesis, Migration

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: PDRG1 interacts with EZH2 and positively regulates EZH2 expression in HCC. (A) IHC staining showing elevated EZH2 expression in HCC tissues compared with adjacent non-tumor tissues (n = 86). (B) Correlation analysis of PDRG1 and EZH2 protein expression in clinical HCC samples. (C) TCGA-LIHC dataset confirming the positive correlation between PDRG1 and EZH2 mRNA expression. (D) Co-IP assays showing direct binding between PDRG1 and EZH2 in HLF cells. (E) Western blot showing that PDRG1 knockdown reduces EZH2 protein levels, whereas EZH2 overexpression does not affect PDRG1 expression. All experiments were performed with three independent biological replicates (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Expressing, Immunohistochemistry, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Knockdown, Over Expression

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: EZH2 restores malignant phenotypes suppressed by PDRG1 knockdown. (A, B) CCK-8 assays showing that EZH2 overexpression restores cell proliferation in PDRG1-silenced HLF and HuH-7 cells. (C, D) Wound-healing assays showing that EZH2 overexpression rescues the migration defects induced by PDRG1 knockdown. (E, F) Transwell assays demonstrating that EZH2 overexpression reverses PDRG1 knockdown-mediated suppression of migration and invasion. (G, H) Colony formation assays showing increased clonogenicity upon EZH2 overexpression in PDRG1-silenced cells. (I, J) EdU assays showing that EZH2 overexpression restores reduced DNA synthesis in PDRG1-knockdown cells. All experiments were performed with three independent biological replicates (n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Knockdown, CCK-8 Assay, Over Expression, Migration, DNA Synthesis

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: PDRG1 regulates p21 transcription through EZH2-mediated H3K27me3 modification. (A) Western blot analysis showing that PDRG1 knockdown decreases EZH2, p-CDK2, and p-RB levels while increasing p21 and SASP factors (IL-1β, IL-6, and IL-8); ectopic EZH2 expression reverses these changes. (B, C) ChIP-qPCR analysis showing that EZH2 knockdown reduces, whereas EZH2 overexpression increases, EZH2 occupancy and H3K27me3 enrichment at the p21 promoter in HLF cells. (D, E) ChIP-qPCR analysis demonstrating that PDRG1 knockdown decreases EZH2 recruitment and H3K27me3 levels at the p21 promoter, both of which are restored by EZH2 re-expression in HLF cells. All experiments were performed with three independent biological replicates (n = 3). * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Modification, Western Blot, Knockdown, Expressing, ChIP-qPCR, Over Expression

Journal: International Journal of Biological Sciences

Article Title: Identification of a Novel PDRG1-EZH2-p21 Pathway Controlling Senescence and Tumor Progression in Hepatocellular Carcinoma

doi: 10.7150/ijbs.129113

Figure Lengend Snippet: Structural identification of the PDRG1-EZH2 binding interfaces and the functional requirement of their interaction in HCC progression. (A) Co-IP using EZH2 and PDRG1 truncation mutants demonstrates that the N-terminal region of EZH2 (amino acids 1-340) and the N-terminal segment of PDRG1 (amino acids 36-70) mediate their interaction in SNU449 cells. (B) Structural modeling predicts a stable interaction interface between the N-terminal helix of PDRG1 and the N-terminal region of EZH2. (C-F) CCK-8, wound-healing, Transwell, and colony formation assays show that overexpression of the PDRG1 Δ36-70 promotes cell proliferation, migration, invasion, and clonogenicity in SNU449 cells. All experiments were performed with three independent biological replicates (n = 3). (G) Western blot analysis indicates that the overexpression of PDRG1 Δ36-70 suppresses p21 and SASP factors and activates p-CDK2 and p-RB in SNU449 cells. (H) Xenograft assays show that SNU449 cells expressing PDRG1 Δ36-70 exhibit enhanced tumor growth, as indicated by increased tumor volume and tumor weight (n = 6). oePDRG1 PD2 : oePDRG1 Δ36-70 , * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Antibodies against DDDDK/Flag (Proteintech, China, 20543-1-AP), HA (Proteintech, China, 51064-2-AP), PDRG1 (Proteintech, China, 16968-1-AP), E-cadherin (Proteintech, China, 20874-1-AP), N-cadherin (Proteintech, China, 22018-1-AP), Vimentin (Proteintech, China, 10366-1-AP), Beta Catenin (Proteintech, China, 51067-2-AP), p21(Proteintech, China, 28248-1-AP), RB (Proteintech, China, 10048-2-Ig), Phospho-RB (Proteintech, China, 84692-1-RR), IL-1 beta (Proteintech, China, 26048-1-AP), IL-6 (Proteintech, China, 21865-1-AP), IL-8 (Proteintech, China, 27095-1-AP) and EZH2 (Proteintech, China, 21800-1-AP) were purchased from Proteintech Technology.

Techniques: Binding Assay, Functional Assay, Co-Immunoprecipitation Assay, CCK-8 Assay, Over Expression, Migration, Western Blot, Expressing