DAND5 Antibody

What is DAND5 and what are its primary biological functions?

DAND5 is an extracellular protein belonging to the Cerberus/DAN family of TGF-β/Nodal signaling antagonists. It functions primarily by binding to Nodal ligands and/or Nodal (co)receptors, preventing agonist-receptor interaction and subsequent signaling activation. DAND5 is considered a multivalent antagonist, as it can also bind to and inhibit BMP and WNT ligands . In developmental contexts, DAND5 plays critical roles in cardiac development, with loss-of-function studies in mice demonstrating increased thickness of ventricular heart walls due to enhanced cardiomyocyte proliferation .

What tissue and cell types commonly express DAND5?

DAND5 expression has been documented in cardiac tissues during development and in pluripotent stem cell-derived cardiomyocytes. Additionally, DAND5 expression has been observed in colorectal cancer tissues, with expression levels that positively correlate with cancer progression . DAND5 protein has also been detected in breast cancer tissues, where its expression correlates with disease outcomes .

How can DAND5 be detected in experimental settings?

DAND5 can be detected through several methodologies depending on the research question:

• Immunohistochemistry (IHC) for tissue expression analysis

• Western blotting for protein expression in cell lysates

• RT-qPCR for mRNA expression analysis

• ELISA for serum concentration measurement in clinical samples

• Immunofluorescence for cellular localization studies

What are the optimal conditions for DAND5 antibody use in Western blotting?

For Western blotting applications, monoclonal anti-DAND5 antibodies have been successfully employed at a dilution of 1:2000 . Protein samples should be separated by SDS-PAGE and transferred to nitrocellulose membranes. After blocking in 5% BSA (w/v) at room temperature for 1 hour, membranes should be incubated with primary anti-DAND5 antibody overnight at 4°C, followed by appropriate secondary antibody incubation for 1 hour at room temperature. Signal detection is typically performed using enhanced chemiluminescence reagents .

How can DAND5 function be studied through gene editing approaches?

CRISPR-Cas9 gene editing technology has been successfully employed to generate DAND5 knockout and correction models. For example, researchers have:

• Generated DAND5-corrected hiPSC lines to serve as isogenic controls

• Created DAND5 full-mutant hiPSC lines to study complete loss of function

• Produced Dand5 knockout mouse embryonic stem cell lines

These gene-edited lines enable the analysis of DAND5's role in processes such as cardiomyocyte differentiation and proliferation by comparing isogenic lines that differ only in DAND5 function .

What approaches are effective for DAND5 knockdown studies?

Transient knockdown of DAND5 can be achieved using siRNA transfection. In published studies, siRNA for DAND5 was synthesized and transfected into cells using TurboFect siRNA Transfection Reagent at a final concentration of 10 nM. Cells were typically collected 48-72 hours post-transfection for subsequent experiments. This approach has been successfully applied in colorectal cancer cell lines such as RKO and HCT116 to study the effects of DAND5 depletion on cell proliferation, migration, and invasion .

How does DAND5 affect cardiomyocyte differentiation from pluripotent stem cells?

Studies using DAND5-deficient pluripotent stem cells have demonstrated that DAND5 inactivation significantly enhances cardiac differentiation. In mouse embryonic stem cells (mESCs), Dand5 knockout resulted in approximately double the number of cardiac beating foci compared to wild-type cells . This enhancement occurs through two main mechanisms:

• Early formation of cardiac progenitor cells, shown by increased proportion of FLK-1+/PDGFR-α+ cardiac progenitor cells

• Increased proliferative capacity of cardiomyocytes derived from DAND5-deficient stem cells

What signaling pathways are affected by DAND5 in cardiomyocyte development?

DAND5 primarily antagonizes the TGF-β/Nodal signaling pathway, with loss of DAND5 function leading to increased levels of phosphorylated-SMAD2, indicating enhanced Nodal signaling activity. Transcriptional profiling of DAND5-deficient cardiomyocytes reveals upregulation of genes involved in:

• Early cardiac gene networks governing differentiation

• Cell cycling pathways

• Cardiac regenerative pathways

Interestingly, genes involved in cardiomyocyte maturation show reduced expression in DAND5-deficient cells, suggesting a complex role in balancing proliferation versus maturation .

What are the functional implications of DAND5 modulation in cardiomyocytes?

Electrophysiological assessments using multi-electrode array (MEA) technology have shown that DAND5-deficient cardiomyocytes, despite their higher proliferative capacity, maintain electrophysiological competence . This suggests that modulating DAND5 levels could potentially serve as a strategy for generating larger numbers of functional cardiomyocytes from pluripotent stem cells for applications such as cardiac repair .

How is DAND5 expression correlated with cancer progression?

In colorectal cancer (CRC), high DAND5 protein expression in tissues increases with advancing TNM stage. Serum DAND5 levels in CRC patients are significantly higher than in normal controls and correlate with:

Similarly, in breast cancer, positive DAND5 staining in tissues is associated with a higher likelihood of disease events (HR=5.494; 95% CI: 1.008-2.353; P=0.048) and poorer disease-free survival (P=0.041) .

What is the potential of serum DAND5 as a biomarker in cancer?

Serum DAND5 has shown promise as a diagnostic and prognostic biomarker in both colorectal and breast cancer:

For CRC diagnosis:

• Cut-off value: 199.5 pg/ml

• High accuracy in distinguishing CRC patients from healthy subjects

• Positive correlation between serum DAND5 levels and matched DAND5 expression in CRC tissues

For breast cancer prognosis:

• Positive serum DAND5 is associated with increased risk of disease events (univariate: HR=1.58; 95% CI: 1.206-2.070; P=0.001)

• Correlates with poor disease-free survival (P=0.001) and distant disease-free survival (P=0.002)

How does DAND5 affect cancer cell behavior in experimental models?

In vitro studies using siRNA-mediated knockdown of DAND5 in colorectal cancer cell lines have demonstrated that DAND5 depletion:

• Significantly decreases cancer cell proliferation

• Reduces cell migration and invasion

• Affects epithelial-mesenchymal transition (EMT) phenotype, likely through modulation of E-cadherin and Snail expression

Additionally, secreted DAND5 has been shown to promote tumor growth and angiogenesis both in vitro and in vivo models of breast cancer .

How should appropriate controls be selected for DAND5 functional studies?

When studying DAND5 function, it is critical to include appropriate controls that isolate the specific effects of DAND5 modulation. The most rigorous approach employs isogenic cell lines that differ only in DAND5 status. Studies have successfully generated:

• DAND5-corrected isogenic control lines from patient-derived iPSCs

• DAND5 full-mutant lines from the same parental cells

• Wild-type versus knockout mESC lines

This approach minimizes confounding variables that might arise from different genetic backgrounds and allows for direct attribution of phenotypic differences to DAND5 function.

What timepoints are critical when evaluating DAND5's effects on cardiomyocyte differentiation?

Studies have shown that DAND5's effects on cardiomyocyte proliferation may vary at different stages of differentiation. For example, in one study, DAND5-variant cardiomyocytes showed significantly increased proliferation at day 10 of differentiation, but this difference diminished by day 20 . Therefore, experimental designs should include multiple timepoints for evaluation, particularly:

• Early differentiation (days 0-5): to capture effects on cardiac progenitor specification

• Mid-differentiation (days 6-12): to evaluate early cardiomyocyte proliferation

• Late differentiation (days 13-20+): to assess maturation and sustained proliferative effects

What methodological approaches best quantify DAND5 effects on cell proliferation?

Multiple complementary techniques should be employed to robustly assess DAND5's effects on cell proliferation:

• Flow cytometry analysis of cell cycle markers

• Colony formation assays (quantifying colonies containing >50 cells)

• Expression analysis of cell cycle regulators via RT-qPCR

• Immunofluorescence staining for proliferation markers (e.g., Ki67, phospho-histone H3)

• EdU incorporation assays to measure DNA synthesis

These approaches collectively provide a comprehensive assessment of proliferative capacity beyond what any single assay could reveal.

How can inconsistencies in DAND5 antibody staining patterns be resolved?

Inconsistent immunostaining results may stem from several factors:

• Antibody specificity: Validate antibodies using positive and negative controls, including DAND5 knockout tissues/cells

• Fixation conditions: Optimize fixation protocols, as DAND5 is an extracellular protein that may require specific preservation methods

• Antigen retrieval: Test multiple antigen retrieval methods if using formalin-fixed, paraffin-embedded samples

• Scoring system standardization: Implement a rigorous scoring system for immunohistochemistry, such as the immunoreactive score used in published studies that multiplies staining intensity (0-3) by percentage of positive cells (0-4)

How should DAND5 serum concentration data be interpreted in clinical studies?

When analyzing DAND5 serum concentration data:

• Establish appropriate cut-off values for the specific cancer type and assay method (e.g., 199.5 pg/ml for CRC, 30.8 pg/ml for breast cancer)

• Use receiver operating characteristic (ROC) curves to determine sensitivity, specificity, and area under the curve (AUC)

• Apply both univariate and multivariate analyses when correlating with clinical outcomes

• Account for potential confounding factors such as tumor stage, grade, and treatment history

• Validate findings in independent cohorts before clinical application

What approaches help resolve contradictory findings regarding DAND5 function?

When encountering contradictory findings about DAND5 function:

• Examine the specific cell types or tissue contexts, as DAND5's effects may be context-dependent

• Consider partial versus complete loss of function (heterozygous variants versus complete knockout)

• Evaluate the efficiency level of DAND5 protein in variant cell lines compared to controls

• Assess the temporal dynamics, as observed effects may change over time

• Analyze the precise molecular mechanisms, as DAND5 can inhibit multiple signaling pathways (Nodal, BMP, WNT)

How might DAND5 modulation be applied in regenerative medicine?

DAND5 modulation shows promise for regenerative medicine applications, particularly for cardiac repair. Since DAND5 inactivation enhances both cardiac progenitor formation and cardiomyocyte proliferation while maintaining functional competence, targeted inhibition of DAND5 could potentially:

• Improve efficiency of deriving cardiomyocytes from patient-specific iPSCs

• Expand cardiomyocyte populations for cell therapy applications

• Enhance cardiac regeneration following injury

• Serve as a target for small molecule modulators to stimulate endogenous cardiac repair

What are the key considerations for developing therapeutic strategies targeting DAND5?

Development of therapeutic approaches targeting DAND5 should consider:

• The multivalent nature of DAND5 inhibition (affects Nodal, BMP, and WNT pathways)

• Potential off-target effects in non-cardiac tissues where DAND5 is expressed

• The balance between proliferation enhancement and proper functional maturation

• Delivery methods for DAND5-targeting agents (e.g., neutralizing antibodies, small molecules, or RNA interference)

• Temporal aspects of intervention, as DAND5's effects may vary at different developmental or disease stages

How can advanced technologies enhance DAND5 structure-function studies?

Emerging technologies that could advance our understanding of DAND5 include:

• Cryo-electron microscopy to elucidate the 3D structure of DAND5 and its interactions with ligands

• Single-cell transcriptomics to characterize cell-specific responses to DAND5 modulation

• CRISPR activation/inhibition systems for precise temporal control of DAND5 expression

• Organ-on-chip models to study DAND5 function in complex tissue contexts

• Computational modeling of DAND5 binding affinities to different signaling molecules