ZNF560 Antibody

What is ZNF560 and what is its primary function in human cells?

ZNF560 (Zinc Finger Protein 560) is a transcriptional regulatory protein belonging to the zinc finger protein family. It is predicted to enable DNA-binding transcription repressor activity and RNA polymerase II-specific transcription regulatory functions. The protein contains zinc finger domains that facilitate nucleic acid binding, allowing it to interact with specific DNA sequences and potentially regulate gene expression. While its exact function is still being elucidated, experimental evidence suggests it plays significant roles in cellular processes related to cell cycle regulation, particularly in pathological contexts such as cancer development .

In which tissues and cell types is ZNF560 typically expressed?

ZNF560 expression has been documented across multiple tissue types, with particularly notable expression in both normal and cancerous tissues. Analysis of TCGA database through UALCAN revealed significant ZNF560 mRNA expression elevation in several human solid tumors, particularly in:

• PRAD (Prostate adenocarcinoma)

• SARC (Sarcoma)

• THCA (Thyroid carcinoma)

• THYM (Thymoma)

The protein shows distinct expression patterns between normal and malignant tissues, with osteosarcoma tissues consistently demonstrating elevated expression levels compared to non-tumoral paired samples . Immunohistochemical studies have confirmed its presence in both nuclear and cytoplasmic regions of osteosarcoma cells .

What are the validated applications for ZNF560 antibodies in research?

ZNF560 antibodies have been validated for multiple experimental applications, though specific validations vary by manufacturer and antibody clone:

Researchers should verify each antibody's specific validation data, as different clones may perform optimally under different conditions .

How does ZNF560 expression correlate with cancer prognosis, particularly in osteosarcoma?

ZNF560 demonstrates significant prognostic value in osteosarcoma. Through comprehensive analysis of both GEO and TCGA databases, researchers have verified increased expression of ZNF560 in osteosarcoma tissues, which strongly correlates with unfavorable clinical outcomes . Specifically:

Similar correlations have been observed in other malignancies, including lung adenocarcinoma (LUAD) and acute myeloid leukemia (AML), indicating ZNF560 may serve as a pan-cancer prognostic biomarker .

What signaling pathways and biological processes are affected by ZNF560 expression or knockdown?

Gene Set Enrichment Analysis (GSEA) comparing high versus low ZNF560 expression groups has revealed significant enrichment in multiple critical cellular pathways:

• Cell cycle regulation pathways

• Cell cycle mitotic processes

• Cell cycle checkpoints

• DNA replication mechanisms

Experimental knockdown of ZNF560 in osteosarcoma cell lines resulted in:

• Decreased expression of cell cycle regulators including PCNA, UBE2C, CDCA5, CDK4, and CDK6

• Significant reduction in cell viability

• Diminished colony formation capability

• Increased apoptosis

• Impaired cell migration and invasion

These findings collectively suggest ZNF560 functions as an oncogene that promotes tumor progression through regulation of cell cycle progression, apoptotic resistance, and enhanced cellular motility .

What experimental approaches have been used to study ZNF560's role in cancer cell migration and invasion?

Researchers have employed multiple complementary techniques to elucidate ZNF560's role in cancer cell migration and invasion:

• Wound healing assay: ZNF560 knockdown via siRNA in both HOS and MG63 osteosarcoma cell lines resulted in profound impairment of cell migration into the wound area compared to control cells transfected with scramble RNA .

• Transwell migration assay: Quantitative analysis demonstrated that the proportion of ZNF560-knockdown cells that migrated through the membrane was significantly lower compared to control cells, confirming ZNF560's essential role in facilitating motility of human osteosarcoma cells .

• Gene expression analysis: Following ZNF560 knockdown, researchers observed downregulation of multiple genes associated with cell motility and metastatic potential, supporting a mechanistic role for ZNF560 in regulating cancer cell invasion .

• Patient-derived xenograft (PDX) models: While not yet fully implemented, researchers have proposed establishing PDX mice models to further elucidate ZNF560's role in tumor formation and metastasis in vivo .

How does ZNF560 interact with other transcription factors and gene regulatory networks?

Comparative gene analysis between high and low ZNF560 expression cohorts has identified several zinc finger proteins with similar expression patterns, including ZNF229 and ZNF728, suggesting potential cooperative or compensatory regulatory networks . While direct protein-protein interactions have not been fully characterized, preliminary evidence suggests ZNF560 may be involved in the transcriptional regulation of the cyclin family .

The zinc finger protein family comprises one of the most prevalent regulatory protein families, with members like ZNF233 and ZNF331 serving as tumor biomarkers in hepatocellular carcinoma and gastric cancer, respectively . ZNF560 likely functions within this broader regulatory network, potentially through:

• Direct binding to promoter regions of target genes

• Interaction with chromatin remodeling complexes

• Competition or cooperation with other transcription factors

• Recruitment of co-activators or co-repressors to specific genomic loci

What protocol is recommended for ZNF560 immunohistochemistry staining?

Based on published research methodologies, the following protocol has been successfully employed for ZNF560 immunohistochemical analysis:

• Tissue preparation:

  • Fix tissues in formalin

  • Embed in paraffin

  • Section at appropriate thickness (typically 4-5 μm)

• Deparaffinization and rehydration:

  • Deparaffinize sections in xylene

  • Rehydrate through graded ethanol series

• Antigen retrieval and blocking:

  • Treat with 3% hydrogen peroxide to inhibit endogenous peroxidase activity

  • Apply appropriate blocking agent to minimize nonspecific binding

• Primary antibody incubation:

  • Incubate overnight at 4°C with anti-ZNF560 primary antibody (1:200 dilution)

  • Several validated antibodies are available (e.g., Biorbyt orb26621)

• Secondary antibody and detection:

  • Incubate with HRP-conjugated goat anti-rabbit IgG (1:200 dilution) at room temperature for 1 hour

  • Incubate with peroxidase-conjugated streptavidin and diaminobenzidine

  • Counterstain with hematoxylin

• Scoring method:

  • Evaluate the proportion of positively stained cells to the nearest 5%

  • Calculate H-scores using the formula: H-score = [1 × (% cells 1+) + 2 × (% cells 2+) + 3 × (% cells 3+)]

  • Compute cytoplasmic, nuclear, and total H-scores (range: 0-300)

  • Use double-blinded scoring for reliability

What strategies are effective for ZNF560 knockdown in cellular models?

Successful ZNF560 knockdown has been achieved using RNA interference approaches:

• siRNA transfection:

  • Researchers have effectively reduced ZNF560 expression using targeted siRNA constructs in both HOS and MG63 osteosarcoma cell lines

  • Confirmation of knockdown efficiency should be performed via Western blotting and qRT-PCR

  • Appropriate scramble RNA controls should be included in all experiments

• shRNA stable knockdown:

  • For long-term studies, genetic strategies such as shRNA constructs have been utilized to create stable ZNF560-knockdown cell lines

  • This approach allows for extended observation of phenotypic changes and functional studies

• Validation of knockdown efficiency:

  • Western blotting confirmation using validated anti-ZNF560 antibodies

  • qRT-PCR analysis of ZNF560 mRNA levels

  • Functional validation through known downstream targets (e.g., PCNA, UBE2C, CDCA5, CDK4, and CDK6)

What controls should be implemented when studying ZNF560 expression?

Rigorous experimental design for ZNF560 studies should incorporate multiple control strategies:

• Expression controls:

  • Positive control tissues/cells with known ZNF560 expression (e.g., osteosarcoma cell lines)

  • Negative control tissues/cells with minimal ZNF560 expression

  • Gradient of expression samples for quantitative studies

• Antibody controls:

  • Isotype control antibodies to assess non-specific binding

  • Secondary antibody-only controls to evaluate background signal

  • Peptide competition assays to confirm antibody specificity

• Knockdown/overexpression controls:

  • Scramble RNA controls for siRNA experiments

  • Empty vector controls for overexpression studies

  • Serial dilution of knockdown/overexpression constructs to establish dose-response relationships

• Data analysis controls:

  • Double-blinded scoring of immunohistochemistry results

  • Independent verification of scoring by multiple researchers

  • Intraclass correlation coefficient (ICC) assessment (>90% recommended for reliable results)

How can researchers address potential non-specific binding issues with ZNF560 antibodies?

When working with ZNF560 antibodies, several strategies can minimize non-specific binding:

• Antibody selection considerations:

  • Use affinity-isolated antibodies when available, as they typically offer higher specificity

  • Polyclonal antibodies against ZNF560 may recognize multiple epitopes, potentially increasing sensitivity but requiring careful validation

• Blocking optimization:

  • Experiment with different blocking agents (BSA, normal serum, commercial blockers)

  • Extend blocking times for challenging samples

  • Consider adding 0.1-0.3% Triton X-100 to reduce hydrophobic interactions

• Dilution optimization:

  • Test multiple antibody dilutions beyond manufacturer recommendations

  • For Western blotting, recommended ranges of 1:500-1:2000 provide starting points

  • For immunofluorescence, 0.25-2 μg/mL concentrations have been validated

• Validation approaches:

  • Confirm specificity using ZNF560-knockdown samples

  • Consider peptide competition assays using the immunogen sequence (available for many commercial antibodies)

What considerations should be made when interpreting ZNF560 expression data across different cancer types?

Researchers analyzing ZNF560 expression across cancer types should consider several important factors:

• Tissue-specific expression patterns:

  • ZNF560 expression varies substantially across tissue types

  • Both nuclear and cytoplasmic localization have been observed, potentially indicating different functions

  • Expression should be compared to appropriate tissue-matched controls

• Methodological variations:

  • Different antibodies may recognize distinct epitopes or isoforms

  • Scoring methods vary between studies (H-score vs. percentage positive vs. intensity)

  • Platform differences (microarray vs. RNA-seq vs. protein-level detection) can affect results

• Data integration challenges:

  • TCGA-SARC datasets have limited normal control samples

  • Complementary datasets (e.g., GSE99671) should be integrated for comprehensive analysis

  • Multivariable Cox regression analysis should complement Kaplan-Meier plots for prognostic evaluation

• Cancer subtype heterogeneity:

  • ZNF560's prognostic significance may vary between cancer subtypes

  • Stratification by histological and molecular subtypes is recommended

  • ZNF560 alterations resulting from chronic hypoxia may influence progression in prostate cancer differently than in osteosarcoma

What emerging research directions are expanding our understanding of ZNF560 function?

Several promising research directions are advancing our understanding of ZNF560 biology:

• Multi-omics integration:

  • Combining transcriptomic, proteomic, and epigenomic data to comprehensively map ZNF560's regulatory network

  • Identifying direct genomic binding sites through ChIP-seq approaches

  • Correlating expression with genome-wide methylation patterns

• Patient-derived models:

  • Development of patient-derived xenograft (PDX) models to study ZNF560's role in tumor formation in vivo

  • Organoid cultures to examine ZNF560 function in three-dimensional tissue architecture

  • Primary patient samples to validate findings from cell line models

• Therapeutic targeting potential:

  • Exploration of ZNF560 as a cancer biomarker for patient stratification

  • Investigation of approaches to modulate ZNF560 expression or activity

  • Analysis of synthetic lethality approaches targeting ZNF560-dependent pathways

• Structural biology approaches:

  • Characterization of ZNF560's DNA-binding domains and specificity

  • Identification of potential protein interaction partners

  • Structure-based design of potential inhibitors targeting ZNF560-mediated transcriptional regulation