CLIC3 Antibody

What is CLIC3 and why is it important in cellular research?

CLIC3 is a chloride intracellular channel protein that plays a crucial role in maintaining cellular homeostasis by regulating the flow of chloride ions across membranes. This regulation is essential for processes such as membrane potential stabilization, signal transduction, and cell volume control, all of which are critical for normal cellular function. CLIC3 is predominantly localized in the nucleus, where it not only stimulates chloride ion channel activity but also interacts with ERK 7, suggesting a potential role in the regulation of cell proliferation . The structure of CLIC3 includes a short hydrophobic domain, indicating that it may require multimerization or interaction with other proteins to function effectively as a membrane channel or channel regulator . CLIC3's diverse expression across various tissues, particularly in the placenta, brain, and heart, underscores its importance in physiological processes and its potential implications in various diseases . These characteristics make CLIC3 an important subject for research in cellular biology, physiology, and pathophysiology.

What applications can CLIC3 antibodies be used for in molecular biology research?

CLIC3 antibodies are versatile tools in molecular biology research with multiple applications. Based on available research data, CLIC3 antibodies can be used for:

• Western Blotting (WB): For detecting CLIC3 protein expression in tissue or cell lysates, with recommended dilutions of 1:1000-1:6000 .

• Immunoprecipitation (IP): To isolate and purify CLIC3 protein complexes for studying protein-protein interactions .

• Immunofluorescence (IF): For visualizing the subcellular localization of CLIC3 in cells, with recommended dilutions of 1:10-1:100 .

• Immunohistochemistry (IHC): To detect CLIC3 in tissue sections, particularly useful for studies involving placenta tissue, with recommended dilutions of 1:50-1:500 .

• Enzyme-linked Immunosorbent Assay (ELISA): For quantitative measurement of CLIC3 protein levels .

These applications enable researchers to investigate CLIC3's expression patterns, localization, interactions, and functional roles in various cellular contexts and experimental conditions.

What types of samples have been successfully tested with CLIC3 antibodies?

CLIC3 antibodies have been successfully tested across a range of biological samples, demonstrating their versatility in research applications. According to the research data, positive results have been confirmed in:

• Cell lines:

  • JAR cells (choriocarcinoma cell line)

  • MCF-7 cells (breast cancer cell line) for immunofluorescence

  • HEK293T cells (transfected with CLIC3) for functional studies

  • Gastric cancer cell lines (MKN7, MKN74, MKN45, KATOIII, and NUGC-4)

• Tissue samples:

  • Human placenta tissue (for both Western blot and immunohistochemistry)

  • Mouse kidney tissue

  • Rat kidney tissue

  • Human gastric cancer tissue samples (used in tissue microarray analysis)

These validated samples provide researchers with confidence when selecting appropriate experimental models for CLIC3-related studies, particularly in cancer research, kidney function investigations, and reproductive biology research focusing on placental tissues .

How should researchers interpret CLIC3 expression in normal versus pathological tissues?

When interpreting CLIC3 expression in normal versus pathological tissues, researchers should consider several important factors. First, establish baseline expression patterns in normal tissues, noting that CLIC3 shows diverse expression across various tissues with particularly high levels in the placenta, brain, and heart . This baseline understanding is crucial before making comparisons to pathological states.

In pathological contexts, such as cancer tissues, researchers should evaluate both the distribution and intensity of CLIC3 staining. In gastric cancer research, for example, a scoring system has been developed where distribution is scored as 0 (0% of total area), 1 (1-50%), or 2 (51-100%), while intensity is scored as 0 (absent), 1 (weak), 2 (moderate), or 3 (strong) . A composite score can then be calculated, with tissues classified as "CLIC3-high" if the sum of distribution and intensity scores is 3 or above .

Additionally, researchers should consider subcellular localization changes. In normal cells, CLIC3 is predominantly localized in the nucleus, but in pathological conditions, altered subcellular distribution may occur, potentially affecting channel function . Functional studies suggest that CLIC3 functions as a Cl− channel in the plasma membrane of gastric cancer cells, and decreased expression affects these cells . This indicates that both expression levels and subcellular localization are important in disease contexts.

For rigorous interpretation, researchers should employ quantitative methods when possible, use appropriate controls, and have evaluations performed independently by multiple researchers who are blinded to the clinical information to prevent bias, as demonstrated in gastric cancer tissue microarray studies .

How can electrophysiological experiments be designed to characterize CLIC3 channel function?

Designing effective electrophysiological experiments to characterize CLIC3 channel function requires careful consideration of multiple technical aspects. Based on established research protocols, the following comprehensive approach is recommended:

• Expression System Selection:

  • Heterologous expression in HEK293T cells has been successfully used for CLIC3 channel characterization, as these cells have minimal endogenous chloride currents .

  • For physiologically relevant studies, use cell lines that naturally express CLIC3, such as MKN7 gastric cancer cells .

• Vector Construction and Transfection:

  • Create expression vectors (e.g., CLIC3-pcDNA4 or CLIC3-pIRES2-AcGFP1) that include tags for detection (such as Xpress-tag) .

  • Use appropriate transfection reagents like PEI-Max according to manufacturer's instructions .

  • For visualization, consider GFP co-expression to identify transfected cells during patch-clamp recordings .

• Patch-Clamp Configuration:

  • Whole-cell patch-clamp recordings provide the most comprehensive assessment of CLIC3 function across the entire cell membrane .

  • Use patch electrodes with a resistance of 2–4 MΩ when filled with pipette solution .

  • Compensate access resistance electrically by approximately 70% to minimize voltage errors .

• Solution Composition:

  • Pipette solution: 140 mM N-methyl-d-glucamine (NMDG), 14 mM HCl, 126 mM l-aspartic acid, 2 mM Na₂ATP, 5 mM MgCl₂, 10 mM HEPES, 1 mM EGTA (pH 7.3 with Tris) .

  • Standard bathing solution: 145 mM NMDG, 145 mM HCl, 7 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES (pH 7.4 with Tris) .

  • Low Cl⁻ bathing solution: 145 mM NMDG, 145 mM l-aspartic acid, 7 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPES (pH 7.4 with Tris) .

• Voltage Protocols:

  • Apply voltage step pulses of 500 ms from -100 to +100 mV in 20-mV increments .

  • Use ramp pulses of 100 ms from -100 to +100 mV to determine reversal potentials quickly .

  • Measure steady-state currents at 450-500 ms on the step pulses .

• Channel Verification:

  • Confirm channel identity by measuring shifts in reversal potential with changes in extracellular Cl⁻ concentration .

  • Test sensitivity to chloride channel blockers such as NPPB .

  • Normalize currents to corresponding membrane capacitance to account for cell size differences .

• Data Acquisition and Analysis:

  • Filter data at 2.9 kHz and digitize at 10 kHz for optimal signal resolution .

  • Use appropriate analysis software (e.g., WinASCD, Clampfit) to analyze current-voltage relationships .

  • Compare results between CLIC3-expressing cells and controls (mock-transfected cells) .

Following this experimental design has revealed that CLIC3 functions as an outwardly rectifying Cl⁻ channel in the plasma membrane, with characteristic electrophysiological properties that can be modulated by chloride concentration and channel blockers .

What is the relationship between CLIC3 expression and cancer progression?

The relationship between CLIC3 expression and cancer progression is complex and appears to be cancer-type specific. In gastric cancer, research has provided significant insights into this relationship through comprehensive tissue microarray (TMA) analysis of tumor specimens from 107 patients . This research evaluated CLIC3 expression levels based on both distribution and intensity of staining, classifying tissues as "CLIC3-high" when the sum of distribution and intensity scores was 3 or above .

Functional studies have demonstrated that CLIC3 operates as a chloride channel in the plasma membrane of gastric cancer cells, and its expression levels appear to influence cellular behavior . Electrophysiological experiments revealed that gastric cancer MKN7 cells endogenously expressing CLIC3 exhibit characteristic chloride currents that are sensitive to the chloride channel blocker NPPB, similar to those observed in cells with exogenously expressed CLIC3 .

Cell proliferation assays further suggest a functional role for CLIC3 in cancer cell growth. In KATOIII and NUGC-4 gastric cancer cell lines, researchers compared proliferation rates between cells transfected with CLIC3-pIRES2-AcGFP1 vector versus empty vector controls (mock) . Conversely, in MKN7 cells that naturally express CLIC3, siRNA-mediated knockdown approaches were used to assess the effects of reducing CLIC3 expression on cell proliferation .

While the detailed results of these proliferation studies are not fully elaborated in the available search results, the experimental design suggests that researchers were investigating whether modulating CLIC3 expression (either increased or decreased) affects cancer cell proliferation rates . This indicates a potential role for CLIC3 in cancer progression mechanisms, possibly through its function as a chloride channel affecting cellular homeostasis, membrane potential, and subsequent signaling pathways that influence cell growth and division.

Future research directions should include comprehensive assessment of CLIC3 expression across larger patient cohorts with detailed clinical outcomes data to establish potential prognostic value, as well as mechanistic studies to elucidate the precise molecular pathways through which CLIC3 may influence cancer progression.

How does CLIC3 interact with other cellular signaling pathways?

CLIC3 interactions with cellular signaling pathways represent a complex area of research with important implications for understanding its physiological and pathological roles. From the available research data, several key interactions and potential signaling connections have been identified:

• ERK7 Interaction:
  CLIC3 has been shown to interact with ERK7 (Extracellular signal-Regulated Kinase 7), suggesting a potential role in the regulation of cell proliferation signaling cascades . This interaction occurs predominantly in the nucleus, where CLIC3 is primarily localized, and may represent a critical connection between chloride channel activity and mitogen-activated protein kinase (MAPK) signaling pathways .

• Membrane Potential and Signal Transduction:
  As a chloride channel, CLIC3 contributes to the regulation of membrane potential, which is a fundamental parameter affecting numerous signaling cascades . Changes in membrane potential can influence voltage-sensitive processes, calcium signaling, and other ion-dependent signaling mechanisms. CLIC3's role in maintaining cellular homeostasis through chloride ion flow regulation therefore places it as a potential modulator of these fundamental signaling processes .

• Cell Volume Regulation Pathways:
  CLIC3's function in cell volume control connects it to osmotic stress response pathways and volume-regulatory signaling cascades . These pathways are critical for cell survival during osmotic challenges and can trigger adaptive responses through various signaling molecules.

• Cancer-Related Signaling:
  In gastric cancer cells, CLIC3 functions as a chloride channel in the plasma membrane, and its expression levels appear to influence cellular behavior, suggesting connections to cancer-related signaling pathways . The experimental approaches used to study CLIC3 in gastric cancer contexts, including siRNA-mediated knockdown and overexpression studies followed by proliferation assays, indicate potential roles in signaling pathways that regulate cell growth and division .

• Multimerization and Protein Interactions:
  CLIC3's structure includes a short hydrophobic domain, indicating that it may require multimerization or interaction with other proteins to function effectively . These protein-protein interactions represent potential points of cross-talk with diverse signaling pathways, though specific interaction partners beyond ERK7 are not fully detailed in the available search results.

Future research should aim to elucidate the complete interactome of CLIC3 using approaches such as co-immunoprecipitation followed by mass spectrometry, proximity labeling techniques, or yeast two-hybrid screening to identify additional signaling proteins that interact with CLIC3. Furthermore, phosphoproteomics analyses following CLIC3 modulation could reveal downstream signaling pathways affected by CLIC3 activity.

What subcellular localization patterns does CLIC3 exhibit and how do they relate to function?

CLIC3 exhibits dynamic subcellular localization patterns that appear to be context-dependent and functionally significant. Understanding these localization patterns provides critical insights into CLIC3's diverse cellular roles:

• Nuclear Localization:
  CLIC3 is predominantly localized in the nucleus under normal conditions . This nuclear localization is significant as it positions CLIC3 to interact with nuclear proteins and potentially influence gene expression or nuclear signaling pathways. Within the nucleus, CLIC3 not only stimulates chloride ion channel activity but also interacts with ERK7, suggesting a role in regulating cell proliferation pathways .

• Plasma Membrane Localization:
  Despite its name as an "intracellular" channel, CLIC3 can also function at the plasma membrane. In exogenous expression systems (CLIC3-expressing HEK293T cells), immunocytochemistry reveals that CLIC3 is expressed partly in the plasma membrane as well as in intracellular compartments . This plasma membrane localization correlates with functional chloride channel activity, as demonstrated by electrophysiological studies showing outwardly rectifying chloride currents in these cells .

• Cancer Cell Localization:
  In gastric cancer cells (MKN7) that endogenously express CLIC3, the protein also functions as a chloride channel at the plasma membrane, as evidenced by electrophysiological recordings showing chloride currents similar to those observed in cells with exogenously expressed CLIC3 . This suggests that in certain pathological contexts, CLIC3 may relocalize or be preferentially expressed at the plasma membrane.

• Tissue-Specific Expression and Localization:
  CLIC3 shows diverse expression patterns across various tissues, with particularly high levels in the placenta, brain, and heart . This tissue-specific expression suggests potentially distinct subcellular localization patterns and functions in different tissue contexts.

• Multimerization and Protein Interactions:
  CLIC3's structure includes a short hydrophobic domain, indicating that it may require multimerization or interaction with other proteins to function effectively as a membrane channel or channel regulator . This characteristic suggests that protein-protein interactions may influence CLIC3's subcellular localization and function.

The relationship between localization and function appears to be bidirectional – CLIC3's localization determines its functional capabilities (nuclear vs. membrane channel functions), while its interactions with other proteins and cellular contexts may drive its localization patterns. This dynamic relationship underscores the importance of studying CLIC3 localization using multiple complementary approaches, including immunocytochemistry, subcellular fractionation, and functional assays in diverse cellular contexts.

What are the optimal protocols for detecting CLIC3 in different experimental contexts?

The optimal protocols for detecting CLIC3 vary based on the experimental context and research objectives. Based on available research data, here are comprehensive protocols for different detection methods:

1. Western Blotting (WB) Protocol:

• Sample Preparation: For membrane proteins, prepare membrane fractions by centrifugation of cell homogenates at 100,000× g for 90 min at 4°C, then resuspend pellets in 250 mM sucrose and 5 mM Tris-HCl (pH 7.4) .

• Protein Loading: Use 30 μg of membrane proteins per lane .

• Denaturation: Treat samples with 2% SDS plus 5% β-mercaptoethanol .

• Antibody Dilution: Use anti-CLIC3 antibody at 1:1000-1:6000 dilution .

• Secondary Antibody: HRP-conjugated anti-rabbit IgG at 1:5000 dilution .

• Detection System: Use enhanced chemiluminescence (ECL) for visualization .

• Quantification: For quantitative analysis, use systems like FujiFilm's LAS-4000 and MultiGauge software .

• Validated Samples: This protocol has been successfully used with JAR cells, human placenta tissue, mouse kidney tissue, and rat kidney tissue .

2. Immunocytochemistry/Immunofluorescence (IF/ICC) Protocol:

• Fixation: Fix cells with ice-cold methanol for 5 min at room temperature .

• Permeabilization: Treat with PBS containing 0.3% Triton X-100 and 0.1% bovine serum albumin (BSA) for 15 min at room temperature .

• Blocking: Block non-specific binding with a solution containing 20 mM phosphate buffer (pH 7.4), 450 mM NaCl, 16.7% goat serum, and 0.3% Triton X-100 .

• Primary Antibody: Incubate with anti-CLIC3 antibody at 1:10-1:100 dilution overnight at 4°C .

• Secondary Antibody: Incubate with fluorophore-conjugated secondary antibodies (e.g., Alexa Fluor 488-conjugated anti-rabbit IgG) at 1:100 dilution for 1 h at room temperature .

• Nuclear Counterstain: Visualize DNA using DAPI (1:1,000) .

• Imaging: Use confocal microscopy (e.g., Zeiss LSM 780) for optimal resolution .

• Validated Samples: Successfully used with MCF-7 cells and transfected HEK293T cells .

3. Immunohistochemistry (IHC) Protocol:

• Sample Preparation: For tissue microarrays (TMA), use 1.0-mm cores of tissues from paraffin-embedded blocks .

• Antigen Retrieval: Use TE buffer pH 9.0 (recommended) or alternatively citrate buffer pH 6.0 .

• Antibody Dilution: Use anti-CLIC3 antibody at 1:50-1:500 dilution .

• Scoring System: For quantitative analysis, evaluate both distribution (0 = 0% of total area, 1 = 1-50%, 2 = 51-100%) and intensity (0 = absent, 1 = weak, 2 = moderate, 3 = strong) of CLIC3 staining .

• Validation: Have two independent researchers evaluate the sections while blinded to clinicopathological information .

• Validated Samples: Successfully used with human placenta tissue .

4. RNA Expression Analysis Protocol:

• RNA Extraction: Extract total RNA using systems like the SV Total RNA Isolation System .

• cDNA Synthesis: Synthesize cDNA using reverse transcriptases such as SuperScript IV according to manufacturer's instructions .

• PCR Amplification: For CLIC3 gene (accession number: NM_004669), use appropriate primers with PCR conditions such as 2 min at 94°C, followed by 50 cycles of 15 s at 94°C, 30 s at 60°C, and 1 min at 68°C .

• Validation: Verify amplified products by sequencing or restriction enzyme analysis.

The choice between these protocols should be guided by the specific research question, available samples, and desired outcomes. For comprehensive characterization, combining multiple detection methods is recommended.

How should researchers troubleshoot common issues with CLIC3 antibody experiments?

When troubleshooting CLIC3 antibody experiments, researchers should adopt a systematic approach to identify and resolve common issues. Based on established research protocols and best practices, here's a comprehensive troubleshooting guide:

1. Western Blotting Issues:

2. Immunocytochemistry/Immunofluorescence Issues:

3. Immunohistochemistry Issues:

4. Functional Assay Issues:

5. General Troubleshooting Approaches:

• Validate antibody specificity using:

  • Positive controls (known CLIC3-expressing samples)

  • Negative controls (CLIC3 knockdown via siRNA)

  • Blocking peptide competition assays

• Optimize experimental conditions:

  • Test multiple antibody dilutions

  • Vary incubation times and temperatures

  • Compare different detection methods

• Verify protein localization:

  • Use subcellular fractionation to confirm nuclear vs. membrane localization

  • Compare results with GFP-tagged CLIC3 localization

• Consider biological variables:

  • Cell cycle stage may affect CLIC3 expression/localization

  • Tissue/cell type differences in CLIC3 expression patterns

By systematically addressing these common issues, researchers can optimize CLIC3 antibody experiments for consistent, reliable results across different experimental contexts.

How can siRNA knockdown experiments be optimized for CLIC3 functional studies?

Optimizing siRNA knockdown experiments for CLIC3 functional studies requires careful consideration of multiple factors to ensure effective silencing and reliable functional assessment. Based on established research protocols, the following comprehensive approach is recommended:

1. siRNA Design and Selection:

• Target Sequence Selection: Use validated siRNA sequences such as CGGACGUGCUGAAGGACUU, which has been successfully employed in CLIC3 knockdown studies .

• Control siRNA: Always include appropriate negative control siRNA with similar GC content but no homology to known genes .

• Visualization Options: Consider using fluorescently labeled siRNA (e.g., Alexa 488-conjugated siRNA) to monitor transfection efficiency .

• Multiple siRNAs: Design and test multiple siRNAs targeting different regions of CLIC3 mRNA to rule out off-target effects.

2. Transfection Optimization:

• Cell Type Considerations: Optimize transfection conditions specifically for your cell line of interest. For gastric cancer MKN7 cells, Lipofectamine 3000 has been successfully used .

• siRNA Concentration: Begin with 20 pmol siRNA per well in a 24-well plate format, and adjust based on knockdown efficiency and cell viability .

• Cell Density: Ensure cells are at appropriate confluence (typically 60-80%) at the time of transfection to maximize efficiency while maintaining cell health.

• Transfection Duration: Allow sufficient time (typically 24-72 hours) for protein turnover and depletion after siRNA transfection before functional assays.

3. Knockdown Verification:

• Western Blotting: Verify CLIC3 protein knockdown using Western blotting of membrane fractions (30 μg protein loading) with anti-CLIC3 antibody (1:1000 dilution) .

• Quantification: Use systems like FujiFilm's LAS-4000 and MultiGauge software to quantify knockdown efficiency .

• Immunofluorescence: Confirm reduced CLIC3 expression at the cellular level using immunofluorescence microscopy.

• Time Course Analysis: Consider performing a time course experiment to determine the optimal time point for maximum knockdown.

4. Functional Assays Following Knockdown:

• Cell Proliferation Analysis:

  • After siRNA transfection, culture cells for 24 hours, then dissociate and replate at defined density (e.g., 1.5 × 10⁴ cells per well in 24-well plates) .

  • Count total cell numbers 48 hours after replating to assess proliferation rates .

  • Compare CLIC3 siRNA-treated cells with negative control siRNA-treated cells.

• Electrophysiology:

  • Perform whole-cell patch-clamp recordings to assess changes in chloride currents following CLIC3 knockdown .

  • Use voltage step protocols (from -100 to +100 mV) and measure steady-state currents .

  • Compare current-voltage relationships between CLIC3 knockdown and control cells.

• Migration and Invasion Assays:

  • Assess whether CLIC3 knockdown affects cell motility and invasive capacity using transwell assays.

  • Quantify the number of migrated/invaded cells and compare between knockdown and control conditions.

5. Rescue Experiments:

• Re-expression of siRNA-resistant CLIC3: To confirm specificity, introduce silent mutations in the CLIC3 cDNA that prevent siRNA binding but maintain the amino acid sequence.

• Functional Rescue: Determine whether re-expression of siRNA-resistant CLIC3 restores the phenotypes observed upon knockdown.

6. Statistical Analysis:

• Perform all experiments in at least triplicate for robust statistical analysis.

• Use appropriate statistical tests to determine the significance of observed differences between knockdown and control conditions.

• Report both the magnitude of knockdown (percentage of protein reduction) and the functional consequences.

By following this optimized protocol, researchers can effectively silence CLIC3 expression and reliably assess its functional roles in cellular processes, particularly in cancer cell models where CLIC3 appears to play significant roles in cellular physiology and pathophysiology .

What considerations are important when selecting cell lines for CLIC3 research?

Selecting appropriate cell lines for CLIC3 research is a critical decision that significantly impacts experimental outcomes and relevance. Based on current research practices, several key considerations should guide this selection process:

1. Endogenous CLIC3 Expression Levels:

Different cell lines express varying levels of endogenous CLIC3, which determines their suitability for specific research questions:

• High CLIC3-Expressing Cell Lines:

  • MKN7 gastric cancer cells show significant endogenous CLIC3 expression, making them ideal for knockdown studies and natural function investigations .

  • JAR choriocarcinoma cells have been validated for CLIC3 expression by Western blot and are suitable for placenta-related research .

  • MCF-7 breast cancer cells have been validated for CLIC3 detection by immunofluorescence .

• Low/No CLIC3-Expressing Cell Lines:

  • HEK293T cells have minimal endogenous CLIC3 expression, making them excellent for overexpression studies and channel characterization .

  • The relative expression levels in other gastric cancer cell lines (MKN74, MKN45, KATOIII, NUGC-4) should be considered when designing comparative studies .

2. Transfection Efficiency and Methodology:

• Cell lines vary significantly in their amenability to different transfection methods:

  • HEK293T cells show high transfection efficiency with PEI-Max, making them ideal for overexpression studies .

  • For gastric cancer cells, specific transfection protocols have been established:

    • KATOIII and NUGC-4 cells can be transfected with expression vectors using PEI-Max .

    • MKN7 cells are effectively transfected with siRNA using Lipofectamine 3000 .

3. Physiological and Pathological Relevance:

• Consider the tissue origin of cell lines in relation to CLIC3's tissue-specific expression patterns:

  • Placenta-derived cell lines may be particularly relevant given CLIC3's high expression in placental tissue .

  • Brain and heart-derived cell lines may also be relevant based on CLIC3's expression profile .

  • Gastric cancer cell lines have established protocols for CLIC3 research and clear disease relevance .

4. Functional Assay Compatibility:

• Different experimental endpoints require cell lines with specific characteristics:

  • For electrophysiology: HEK293T cells are well-suited due to their large size, regular morphology, and established patch-clamp protocols .

  • For proliferation assays: KATOIII, NUGC-4, and MKN7 cells have validated protocols .

  • For subcellular localization studies: MCF-7 cells have been validated for immunofluorescence detection of CLIC3 .

5. Genetic Background Considerations:

• Consider the mutation status and genetic alterations in potential cell lines:

  • For cancer studies, select cell lines that represent the molecular subtypes of interest.

  • Consider whether the presence of other mutations might confound CLIC3 functional studies.

  • When possible, use matched cell line pairs (e.g., parent and CLIC3-knockout/overexpressing derivatives).

6. Practical Experimental Factors:

• Growth characteristics and culture requirements

  • Doubling time affects experimental timeline planning

  • Media requirements and supplements needed

  • Adherent vs. suspension growth patterns

• Availability of supporting research data

  • Availability of genomic, transcriptomic, and proteomic data

  • Previous CLIC3-related findings in specific cell lines

7. Complementary Approaches:

• Consider using multiple cell lines with different characteristics to strengthen research findings:

  • Pair endogenous expression models with overexpression systems

  • Include both normal and cancer-derived cell lines when relevant

  • Validate key findings across multiple cell lineages

By carefully evaluating these considerations, researchers can select optimal cell line models for CLIC3 research that align with their specific research questions and experimental approaches, ultimately enhancing the reliability and translational relevance of their findings.

How can researchers verify the specificity of CLIC3 antibodies in their experimental systems?

Verifying antibody specificity is crucial for generating reliable and reproducible results in CLIC3 research. Based on established research practices, here is a comprehensive approach to verifying CLIC3 antibody specificity:

1. Positive Control Validation:

• Use known CLIC3-expressing samples as positive controls:

  • Validated cell lines: JAR cells, MCF-7 cells, MKN7 cells

  • Validated tissues: Human placenta tissue, mouse kidney tissue, rat kidney tissue

  • Overexpression systems: HEK293T cells transfected with CLIC3-pcDNA4 or CLIC3-pIRES2-AcGFP1 vectors

2. Genetic Knockdown/Knockout Controls:

• Perform siRNA-mediated knockdown experiments:

  • Use validated siRNA sequences (e.g., CGGACGUGCUGAAGGACUU) to reduce CLIC3 expression

  • Compare antibody signals between control and CLIC3-depleted samples

  • A specific antibody should show significantly reduced signal in knockdown samples

• CRISPR/Cas9 knockout validation:

  • Generate CLIC3 knockout cell lines as negative controls

  • Antibody should show no signal in complete knockout cells

3. Tagged-Protein Co-localization:

• Express tagged versions of CLIC3:

  • Utilize Xpress-tagged CLIC3 with anti-Xpress antibodies

  • Compare localization patterns detected by anti-CLIC3 and anti-tag antibodies

  • Co-localization confirms specificity of the CLIC3 antibody

4. Peptide Competition Assays:

• Pre-incubate the CLIC3 antibody with excess immunizing peptide

• Perform side-by-side comparisons with and without peptide competition

• Specific signals should be blocked by the competing peptide

5. Western Blot Validation:

• Verify correct molecular weight:

  • CLIC3 should appear as a single band of expected molecular weight

  • Compare with positive controls expressing known levels of CLIC3

  • Look for consistency across different sample types

• Multiple antibody validation:

  • Test different CLIC3 antibodies targeting distinct epitopes

  • Consistent results across antibodies strengthen specificity claims

6. Cross-Species Reactivity Assessment:

• Test the antibody across species of interest:

  • Many CLIC3 antibodies detect mouse, rat, and human CLIC3

  • Confirm specificity in each species independently

  • Consider species-specific differences in CLIC3 expression patterns

7. Multi-technique Confirmation:

• Validate specificity across multiple detection methods:

  • Western blotting (WB): Look for single bands of correct size

  • Immunofluorescence (IF): Compare subcellular localization patterns with literature

  • Immunohistochemistry (IHC): Evaluate tissue distribution consistent with known expression

  • Immunoprecipitation (IP): Confirm ability to pull down CLIC3 specifically

8. Functional Correlation:

• Correlate antibody detection with functional measures:

  • In CLIC3 overexpressing cells, higher antibody signal should correlate with increased chloride channel activity

  • In CLIC3 knockdown cells, reduced antibody signal should correlate with decreased channel function

9. Rigorous Controls in Each Experiment:

• No primary antibody controls

• Isotype controls (using non-specific IgG of the same species)

• Secondary antibody-only controls

• Concentration-matched controls for blocking experiments

10. Documentation and Reporting:

• Record complete antibody information:

  • Catalog number (e.g., sc-390006, 15971-1-AP)

  • Clone designation (e.g., D-11)

  • Lot number and source

  • Validation methods employed

  • Optimized dilutions for each application (WB: 1:1000-1:6000; IHC: 1:50-1:500; IF: 1:10-1:100)

By implementing this comprehensive validation strategy, researchers can establish high confidence in the specificity of their CLIC3 antibodies, ensuring that experimental observations truly reflect CLIC3 biology rather than non-specific artifacts or cross-reactivity.

What are common pitfalls in interpreting CLIC3 functional data and how can they be avoided?

1. Overinterpreting Correlation as Causation:

Pitfall: Assuming that correlations between CLIC3 expression and cellular phenotypes (such as proliferation rates in cancer cells) indicate direct causation.

Solution:

• Perform rescue experiments by reintroducing CLIC3 after knockdown to confirm specificity

• Use multiple approaches to modulate CLIC3 (siRNA, CRISPR, pharmacological inhibitors)

• Establish clear mechanistic links between CLIC3 and observed phenotypes through pathway analysis

2. Neglecting Channel vs. Non-Channel Functions:

Pitfall: Focusing exclusively on CLIC3's chloride channel functionality while overlooking potential non-channel roles.

Solution:

• Design experiments that distinguish between ion conductance and other functions

• Create channel-dead mutants (mutations in the putative pore region) that retain protein-protein interaction capabilities

• Consider CLIC3's nuclear localization and ERK7 interaction when interpreting functional data

3. Misattributing Current Identity in Electrophysiology:

Pitfall: Incorrectly identifying currents as CLIC3-mediated without proper controls and verification.

Solution:

• Confirm current identity through multiple approaches:

  • Verify ion selectivity through reversal potential shifts with altered ionic compositions

  • Test sensitivity to known chloride channel blockers like NPPB

  • Compare currents between CLIC3-expressing and control cells

  • Normalize currents to membrane capacitance to account for cell size differences

4. Ignoring Cell Type-Specific Behaviors:

Pitfall: Generalizing CLIC3 function across cell types without considering context-specific behaviors.

Solution:

• Validate key findings across multiple cell types

• Consider tissue-specific expression patterns (placenta, brain, heart have high CLIC3 expression)

• Account for differences in endogenous CLIC3 levels between cell lines

• Interpret results within the appropriate cellular context (normal vs. cancer cells)

5. Overlooking Subcellular Localization:

Pitfall: Failing to consider how CLIC3's subcellular distribution affects its function.

Solution:

• Perform careful subcellular fractionation to determine protein localization

• Use immunocytochemistry to visualize CLIC3 distribution patterns

• Consider that CLIC3 can localize to both nuclear and plasma membrane compartments with potentially different functions

• Track changes in localization upon experimental manipulations

6. Inadequate Quantification Methods:

Pitfall: Using subjective or non-standardized methods to quantify CLIC3 expression or function.

Solution:

• Implement standardized scoring systems for tissue samples (e.g., combined distribution and intensity scores)

• Have multiple blinded observers evaluate samples to prevent bias

• Use objective measurements from calibrated instruments (e.g., FujiFilm's LAS-4000 system and MultiGauge software for Western blots)

• Report both raw data and normalized values

7. Confounding by Off-Target Effects:

Pitfall: Attributing phenotypes to CLIC3 modulation when they actually result from off-target effects.

Solution:

• Use multiple siRNA sequences targeting different regions of CLIC3 mRNA

• Include appropriate negative controls (scrambled siRNA)

• Verify knockdown specificity through Western blotting or qPCR

• Consider potential compensation by other CLIC family members

8. Oversimplifying Multimerization and Interactions:

Pitfall: Ignoring CLIC3's potential to form multimers or interact with other proteins.

Solution:

• Consider that CLIC3's short hydrophobic domain suggests it may require multimerization or interaction with other proteins to function effectively

• Assess protein-protein interactions through co-immunoprecipitation or proximity labeling

• Evaluate potential heteromeric interactions with other channel proteins

9. Neglecting Temporal Dynamics:

Pitfall: Failing to consider time-dependent changes in CLIC3 expression or function.

Solution:

• Perform time-course experiments to capture dynamic changes

• Consider cell cycle effects on CLIC3 expression and function

• Allow sufficient time after experimental manipulations before functional assessment

Solution:

• Ensure adequate statistical power through appropriate sample sizes

• Validate key findings through independent experimental approaches

• Use tissue microarrays with sufficient patient samples (e.g., 107 gastric cancer patients)

• Report confidence intervals along with statistical significance

What are the most promising future directions for CLIC3 research?

The current state of CLIC3 research reveals several exciting and promising directions for future investigation. Based on the established findings and emerging questions in the field, the following research directions hold particular promise:

• Therapeutic Targeting in Cancer:
  The demonstrated roles of CLIC3 in gastric cancer cell proliferation and its function as a chloride channel in cancer cell membranes suggest significant therapeutic potential . Future research should explore small molecule inhibitors specific to CLIC3 and evaluate their efficacy in preclinical cancer models. Additionally, investigating CLIC3's roles across multiple cancer types beyond gastric cancer could reveal broader therapeutic applications.

• Structural Biology and Channel Gating Mechanisms:
  Further investigation into CLIC3's structure, particularly its short hydrophobic domain and its potential requirement for multimerization or interaction with other proteins to function effectively as a membrane channel, would provide crucial insights into its working mechanism . Cryo-EM or crystallography studies of CLIC3 in different conformational states would enhance our understanding of its channel properties and facilitate rational drug design.

• Signaling Network Integration:
  CLIC3's interaction with ERK7 suggests important connections with signaling networks controlling cell proliferation . Comprehensive interactome studies using proximity labeling or mass spectrometry approaches could reveal the full extent of CLIC3's protein-protein interaction network and its position within cellular signaling cascades, potentially uncovering new therapeutic targets and biological functions.

• Tissue-Specific Functions:
  The diverse expression of CLIC3 across tissues, particularly in the placenta, brain, and heart, suggests important tissue-specific functions that remain largely unexplored . Tissue-specific knockout models could reveal these specialized roles and potential connections to developmental processes or tissue-specific pathologies.

• Subcellular Dynamics and Trafficking:
  The dual localization of CLIC3 in both nuclear and membrane compartments raises important questions about its trafficking and regulation . Live-cell imaging studies with fluorescently tagged CLIC3 could reveal its dynamic movement between compartments and the regulatory mechanisms controlling its localization in response to cellular signals or stresses.

• Translational Biomarker Development:
  Building on the tissue microarray studies in gastric cancer , further investigation into CLIC3 as a potential diagnostic or prognostic biomarker across multiple diseases represents an important translational direction. Large-scale patient cohort studies with long-term follow-up could establish the clinical utility of CLIC3 expression patterns in disease management.

• Physiological Regulation of Ion Homeostasis:
  Expanding the electrophysiological characterization of CLIC3 to understand its contribution to cellular chloride homeostasis under various physiological and pathological conditions would provide important insights into its fundamental biological roles . This could include investigating CLIC3's response to cellular stresses, volume changes, or inflammatory signals.

• CLIC Family Functional Redundancy and Cooperation:
  Comparative studies between CLIC3 and other CLIC family members could reveal functional redundancies, compensatory mechanisms, or cooperative behaviors that influence cellular physiology and disease processes. This could include studying combined knockdown models or investigating hetero-oligomeric interactions between family members.

• Regulation of CLIC3 Expression and Activity:
  Investigating the transcriptional, post-transcriptional, and post-translational mechanisms that regulate CLIC3 expression and activity would provide important insights into how its function is controlled in different cellular contexts. This could include studies of promoter regulation, RNA stability, or post-translational modifications affecting channel function.

• Development of Improved Research Tools:
  Creating more specific antibodies, function-blocking nanobodies, or optogenetic tools to modulate CLIC3 activity with higher temporal and spatial precision would enable more sophisticated functional studies. Additionally, developing CLIC3-specific fluorescent probes or biosensors would enable real-time monitoring of its activity in living cells.

These future directions represent exciting opportunities to expand our understanding of CLIC3 biology and potentially translate these insights into clinical applications. The multifaceted nature of CLIC3—functioning both as an ion channel and potentially as a signaling protein, with diverse tissue expression and subcellular localizations—ensures that its continued investigation will yield important discoveries across multiple areas of cell biology and medicine.

How does understanding CLIC3 contribute to broader knowledge in cell biology and disease mechanisms?

Understanding CLIC3 provides significant contributions to broader knowledge in cell biology and disease mechanisms through multiple interconnected pathways. The unique properties and functions of CLIC3 offer insights that extend well beyond this specific protein, illuminating fundamental biological processes and disease mechanisms:

1. Ion Channel Biology and Membrane Dynamics:

CLIC3's function as an outwardly rectifying chloride channel provides important insights into ion homeostasis regulation . Unlike conventional multi-pass transmembrane channels, CLIC3 represents a distinct class of ion channels that can transition between soluble and membrane-integrated states . This unusual property challenges traditional models of ion channel structure and function, expanding our understanding of membrane protein dynamics. The electrophysiological characterization of CLIC3 reveals how ion channels can exhibit complex gating behaviors and contribute to cellular electrical properties through outward rectification , enriching our understanding of bioelectrical signaling beyond classical models.

2. Subcellular Compartmentalization and Protein Moonlighting:

CLIC3's presence in both nuclear and plasma membrane compartments exemplifies the concept of protein moonlighting, where a single protein performs distinct functions in different cellular locations . This dual localization pattern contributes to our understanding of how cells maximize functional diversity with a limited proteome. The nuclear functions of CLIC3, potentially involving ERK7 interaction, alongside its membrane channel activity, demonstrate how subcellular targeting can dramatically alter protein function . This provides a powerful model for studying how cellular compartmentalization creates functional diversity and regulatory complexity.

3. Cancer Biology and Proliferation Control:

Research on CLIC3 in gastric cancer has revealed connections between ion channel activity and cancer cell proliferation, highlighting the often-overlooked role of ion transport in oncogenesis . The tissue microarray studies demonstrating variable CLIC3 expression across tumor samples exemplify how molecular heterogeneity within cancer can be systematically analyzed . The experimental approaches used to study CLIC3's effects on cancer cell proliferation provide methodological frameworks for investigating other potential cancer-associated proteins . These insights contribute to the emerging field of ion channel oncology, which recognizes ion channels as potential therapeutic targets rather than just passive cellular components.

4. Signaling Pathway Integration:

CLIC3's interaction with ERK7 suggests important connections between ion transport mechanisms and kinase signaling cascades . This intersection of traditionally separate signaling modalities (ion flux and protein phosphorylation) exemplifies how cells integrate diverse signaling languages. Understanding such integrative nodes helps explain how cells process complex information and make coordinated responses to environmental changes. CLIC3 thus serves as a model for studying signaling network connectivity and cross-talk between different cellular communication systems.

5. Protein Structure-Function Relationships:

The structural features of CLIC3, particularly its short hydrophobic domain and potential requirement for multimerization or interaction with other proteins to function effectively, highlight important principles in protein structure-function relationships . These characteristics demonstrate how proteins can dynamically respond to their environment through conformational changes or complex formation. Understanding CLIC3's structural requirements for channel formation provides insights into how proteins can transition between soluble and membrane-integrated states, a fundamental question in cell biology.

6. Translational Research Models:

7. Therapeutic Target Identification:

The functional characterization of CLIC3 in disease contexts establishes a pathway for identifying and validating novel therapeutic targets . The demonstration that CLIC3 functions as a chloride channel that can be blocked by compounds like NPPB provides proof-of-concept for pharmacological intervention . This research paradigm—moving from molecular characterization to functional validation to potential therapeutic targeting—serves as a template for drug discovery efforts focused on other unconventional targets. CLIC3 research thus contributes to expanding the druggable proteome beyond traditional target classes.

8. Cellular Homeostasis and Stress Response:

CLIC3's role in cell volume control and membrane potential stabilization connects to broader understanding of how cells maintain homeostasis under varying conditions . Ion transport processes represent fundamental mechanisms for adapting to environmental changes and stresses. CLIC3's diverse tissue expression, particularly in critical organs like the brain and heart, suggests roles in specialized homeostatic processes in these tissues . These connections illuminate the intricate regulatory networks that maintain cellular and tissue function despite environmental challenges.