FAM110C Antibody

What is FAM110C and what are its known biological functions?

FAM110C (Family with Sequence Similarity 110 Member C) is a protein-coding gene that has been implicated in several critical cellular processes. Recent studies have identified FAM110C as a potential biomarker in various cancers, particularly glioblastoma (GBM) and pancreatic ductal adenocarcinoma (PDAC). In wild-type GBM, FAM110C has been shown to contribute to cell growth and migration . Interestingly, the expression levels of FAM110C differ significantly between IDH1 mutant and wild-type GBM, with higher expression observed in the wild-type variant . In contrast, in pancreatic cancer, FAM110C has been identified as a potential tumor suppressor that can activate ATM and NHEJ signaling pathways through its interaction with HMGB1 . This dual role highlights the context-dependent function of FAM110C across different cancer types.

What research applications are FAM110C antibodies typically used for?

FAM110C antibodies are primarily utilized in several key research applications:

• Western blot (WB) analysis to detect and quantify FAM110C protein expression in cell lysates and tissue samples

• Immunohistochemistry (IHC) to visualize FAM110C expression patterns in tissue sections

• Immunoprecipitation (IP) to isolate FAM110C and study its protein-protein interactions

• Immunofluorescence (IF) to determine the subcellular localization of FAM110C

These applications are critical for investigating FAM110C's role in disease progression, particularly in cancer research where expression levels may correlate with prognosis and treatment response .

How should researchers validate the specificity of FAM110C antibodies?

Proper validation of FAM110C antibodies is essential for obtaining reliable experimental results. Recommended validation approaches include:

• Positive and negative controls: Use cell lines with known FAM110C expression levels. Based on published data, MIAPaCa-2 and JF-305 cells show minimal expression, while Panc3.11, Panc5.04, and Panc10.05 cells demonstrate higher expression levels .

• siRNA/shRNA knockdown: Compare FAM110C detection in cells with and without gene silencing.

• Overexpression validation: Test the antibody in cells transfected with FAM110C expression vectors, similar to the approach described in the literature using pCDH-CMV-MCS-puro plasmid .

• Multiple antibody comparison: Use antibodies that recognize different epitopes of FAM110C to confirm specificity.

• Peptide competition assays: Pre-incubate the antibody with the immunizing peptide to confirm signal specificity.

These validation steps are crucial for ensuring that experimental observations truly reflect FAM110C biology rather than antibody cross-reactivity.

What expression patterns of FAM110C have been observed across different cell types?

Expression patterns of FAM110C vary significantly across different cell types and disease states:

This expression pattern data is valuable for selecting appropriate positive and negative controls when validating antibodies and designing experiments .

How does FAM110C expression correlate with cancer progression and patient outcomes?

FAM110C expression and its epigenetic regulation show significant correlations with cancer progression and patient outcomes:

In GBM:

• FAM110C is highly expressed in wild-type GBM compared to IDH1-mutant GBM (P = 0.0053)

• Higher FAM110C expression correlates with poorer prognosis in wild-type GBM patients (P = 0.03, HR = 1.55 [95% CI 1.04, 2.31])

• FAM110C expression can predict survival in wild-type GBM patients at 1, 3, and 5 years (ROC-1 year: 0.647; ROC-3 years: 0.709; ROC-5 years: 0.932)

• Expression increases with tumor grade (P = 6.7E-05)

In pancreatic lesions:

These findings highlight the potential value of FAM110C as a diagnostic and prognostic biomarker in multiple cancer types .

What signaling pathways are affected by FAM110C expression or silencing?

FAM110C interacts with several important signaling pathways that influence cancer cell behavior:

In GBM:

• Gene Set Enrichment Analysis (GSEA) revealed that FAM110C expression is associated with:

  • Cytokine-receptor interactions

  • Chemokine signaling pathways

  • Cell adhesion molecule pathways

• These pathways align with the observed role of FAM110C in promoting glioma cell migration and invasion

In PDAC:

• FAM110C activates ATM and Non-Homologous End Joining (NHEJ) signaling pathways by interacting with HMGB1

• Loss of FAM110C sensitizes PDAC cells to DNA damage response inhibitors:

  • VE-822 (an ATR inhibitor)

  • MK-8776 (a CHK1 inhibitor)

These differential pathway interactions may explain the context-dependent roles of FAM110C in different cancer types and suggest potential therapeutic strategies targeting these pathways .

How can researchers investigate FAM110C methylation status and its functional consequences?

To investigate FAM110C methylation status and its functional consequences, researchers should consider the following methodological approaches:

• Methylation status assessment:

  • Methylation-specific PCR (MSP) as used in the referenced studies

  • Bisulfite sequencing (BSSQ) for validating methylation density

  • 5-aza-2'-deoxycytidine treatment to confirm epigenetic regulation

• Functional consequences investigation:

  • Re-expression experiments in methylated cell lines using expression vectors

  • RNA interference to silence FAM110C in unmethylated cell lines

  • Phenotypic assays (proliferation, migration, apoptosis) to assess functional impact

  • Cell cycle analysis using flow cytometry following synchronization by serum withdrawal

• Correlation with clinical parameters:

  • Analyze methylation status in patient samples alongside clinical data

  • Perform survival analysis stratified by methylation status

  • Correlate with tumor characteristics (size, stage, differentiation)

This multi-layered approach provides comprehensive insights into how FAM110C methylation affects cellular function and patient outcomes .

What is the relationship between FAM110C expression and sensitivity to DNA damage response inhibitors?

Research has uncovered a synthetic lethal relationship between FAM110C expression and sensitivity to DNA damage response (DDR) inhibitors:

• Loss of FAM110C expression sensitizes PDAC cells to:

  • VE-822 (an ATR inhibitor)

  • MK-8776 (a CHK1 inhibitor)

• The mechanistic basis for this synthetic lethality involves:

  • FAM110C's role in activating ATM and NHEJ signaling pathways through HMGB1 interaction

  • When FAM110C is silenced (by methylation or other mechanisms), cells become more dependent on ATR/CHK1 pathways for DNA damage repair

  • Inhibiting these compensatory pathways with targeted inhibitors leads to synthetic lethality

This relationship suggests a potential therapeutic strategy:

• Identify patients with FAM110C methylation/silencing

• Target these tumors with ATR/CHK1 inhibitors

• Exploit the synthetic lethal interaction for improved therapeutic efficacy

For experimental validation, researchers can use half-inhibitory concentration analysis with MTT assays after treating cells with gradient dilutions of inhibitors .

What are the optimal protocols for detecting FAM110C via Western blot?

For optimal detection of FAM110C via Western blot, researchers should consider the following protocol recommendations:

• Sample preparation:

  • Extract total protein from cells using RIPA buffer with protease inhibitors

  • Determine protein concentration using BCA or Bradford assay

  • Load 20-40 μg of protein per lane for cell lysates

• Gel electrophoresis and transfer:

  • Use 10-12% SDS-PAGE gels (FAM110C is approximately 33 kDa)

  • Transfer to PVDF membrane at 100V for 90 minutes in cold transfer buffer

• Antibody selection and dilution:

  • Primary antibody: Use validated anti-FAM110C antibodies (available commercially)

  • Typical dilution range: 1:500 to 1:1000 for primary antibody

  • Secondary antibody: HRP-conjugated anti-species IgG at 1:5000 dilution

• Controls and validation:

  • Positive control: Lysate from cells with confirmed FAM110C expression (e.g., Panc3.11, Panc5.04, Panc10.05)

  • Negative control: Lysate from cells without FAM110C expression (e.g., MIAPaCa-2, JF-305)

  • Loading control: β-actin or GAPDH

• Detection:

  • Use enhanced chemiluminescence (ECL) detection reagents

  • Optimize exposure time to avoid signal saturation

Following these guidelines will help ensure specific and reliable detection of FAM110C protein in experimental samples .

How should researchers design experiments to investigate FAM110C's role in cell migration?

To effectively investigate FAM110C's role in cell migration, researchers should design comprehensive experiments based on established protocols:

• Experimental models:

  • Cell line selection: Use paired models with differential FAM110C expression

  • For GBM studies: Ln229 and U87 cell lines are appropriate models

  • For PDAC studies: Panc10.05 (high expression) and MIAPaCa-2 (low expression) are suitable

• Manipulation of FAM110C expression:

  • Knockdown: siRNA or shRNA targeting FAM110C in high-expressing cells

  • Overexpression: Transfection with expression vectors in low-expressing cells

  • Validation: Confirm altered expression by qRT-PCR and Western blot

• Migration assays:

  • Transwell migration assay: Assess directional cell migration through membrane

  • Wound healing assay: Evaluate collective cell migration by creating a "scratch"

  • Time-lapse microscopy: Monitor cell movement in real-time

• Analysis and quantification:

  • For transwell assays: Count migrated cells in multiple fields

  • For wound healing: Measure wound closure over time

  • Statistical analysis: Compare migration between FAM110C-manipulated and control cells

• Pathway analysis:

  • Assess effects on signaling pathways identified in GSEA analysis:

    • Cytokine-receptor interactions

    • Chemokine signaling

    • Cell adhesion molecules

  • Validate pathway alterations through Western blot of key signaling proteins

This experimental approach will provide comprehensive insights into FAM110C's functional role in cell migration .

What experimental approaches can reveal FAM110C's protein interactions?

To investigate FAM110C's protein interactions, researchers should employ multiple complementary approaches:

• Co-immunoprecipitation (Co-IP):

  • Use anti-FAM110C antibodies to pull down protein complexes

  • Identify interacting partners through Western blot or mass spectrometry

  • Confirm specificity with IgG control and reverse Co-IP

  • This approach successfully identified the FAM110C-HMGB1 interaction

• Proximity ligation assay (PLA):

  • Visualize protein-protein interactions in situ

  • Provides spatial information about where interactions occur within cells

  • Use specific antibodies against FAM110C and potential interacting partners

• Bimolecular fluorescence complementation (BiFC):

  • Express FAM110C and candidate interactors as fusion proteins with split fluorescent protein fragments

  • Interaction brings fragments together, restoring fluorescence

  • Enables live-cell visualization of interactions

• Yeast two-hybrid screening:

  • Systematic approach to identify novel interacting partners

  • Use FAM110C as bait to screen a library of prey proteins

  • Validate hits with orthogonal methods

• Bioinformatic analysis:

  • Predict potential interactions based on protein domains and motifs

  • Use STRING, BioGRID, or other interaction databases to guide experimental design

  • Focus on proteins involved in ATM and NHEJ pathways, given FAM110C's established role

These approaches provide complementary data on FAM110C's interactome, offering insights into its molecular functions and mechanism of action .

How can researchers use FAM110C expression for patient stratification in clinical studies?

Researchers can utilize FAM110C expression or methylation status for patient stratification in clinical studies using the following approaches:

What are promising therapeutic strategies targeting FAM110C-related pathways?

Several promising therapeutic strategies targeting FAM110C-related pathways warrant further investigation:

• Synthetic lethality approaches:

  • Targeting ATR/CHK1 inhibitors in FAM110C-methylated cancers

  • The small molecules VE-822 (ATR inhibitor) and MK-8776 (CHK1 inhibitor) have shown efficacy in FAM110C-silenced cells

  • Developing combination therapies that enhance this synthetic lethal interaction

• Methylation-based therapies:

  • In cancers where FAM110C acts as a tumor suppressor (PDAC), demethylating agents could restore expression

  • Testing combinations of demethylating agents with conventional chemotherapy

• Small molecule inhibitors:

  • The Connectivity Map (CMap) analysis identified felibinac and fludrocortisone as potential targeted drugs for FAM110C in wild-type GBM

  • These compounds showed mean connective scores of −0.554 and −0.561, respectively (P = 0.001)

  • Further validation and optimization of these compounds could yield effective therapies

• Pathway-based approaches:

  • Targeting cytokine-receptor interactions and chemokine signaling pathways in GBM where FAM110C promotes migration

  • Developing inhibitors of HMGB1-FAM110C interaction in cancers where this interaction is oncogenic

• Biomarker-guided therapy selection:

  • Using FAM110C expression or methylation status to guide therapy choices

  • Personalizing treatment based on predicted sensitivity to ATR/CHK1 inhibitors

These approaches represent promising avenues for translating FAM110C research into clinical applications .

What technological advances would enhance FAM110C research?

Several technological advances would significantly enhance FAM110C research and accelerate discoveries:

• Improved antibody development:

  • Generation of monoclonal antibodies with higher specificity for different FAM110C isoforms

  • Development of phospho-specific antibodies to detect post-translational modifications

  • Creation of antibodies suitable for chromatin immunoprecipitation (ChIP) applications

• Advanced imaging techniques:

  • Super-resolution microscopy to visualize FAM110C localization at nanoscale resolution

  • Live-cell imaging systems to track FAM110C dynamics during cell cycle and migration

  • Multiplexed imaging to simultaneously visualize FAM110C and its interaction partners

• Single-cell analysis technologies:

  • Single-cell RNA-seq to characterize heterogeneity in FAM110C expression within tumors

  • Single-cell proteomics to quantify FAM110C protein levels at cellular resolution

  • Spatial transcriptomics to map FAM110C expression patterns within tissue architecture

• CRISPR-based functional genomics:

  • CRISPR activation/inhibition systems for precise modulation of FAM110C expression

  • CRISPR screens to identify synthetic lethal interactions with FAM110C

  • Base editing to introduce or correct specific mutations in FAM110C

• Computational approaches:

  • Machine learning algorithms to predict FAM110C interactions and functional effects

  • Integrative multi-omics analysis to place FAM110C in broader cellular networks

  • Structural biology predictions to model FAM110C protein structure and interactions

These technological advances would address current limitations in FAM110C research and facilitate more comprehensive understanding of its biological functions and clinical applications.