FABP9 Antibody

What is FABP9 and what is its significance in prostate cancer research?

FABP9 is a member of the fatty acid-binding protein family involved in fatty acid transport and metabolism. In prostate cancer research, FABP9 has emerged as a valuable prognostic marker. Studies have shown that FABP9 protein is highly expressed in highly malignant prostate cancer cell lines (PC-3 and PC3-M), while its expression in benign PNT-2 and some other malignant cell lines is undetectable . Immunohistochemical analyses reveal significantly higher FABP9 staining intensity in carcinoma tissues compared to benign prostatic hyperplasia (BPH) tissues, with increased expression correlating with reduced patient survival times . Furthermore, suppression of FABP9 expression in highly malignant PC3-M cells has been shown to inhibit their invasive potential, suggesting an important role in cancer progression .

How is FABP9 expression detected in tissue samples?

The primary method for detecting FABP9 expression in tissue samples is immunohistochemical (IHC) staining. Typically, formalin-fixed, paraffin-embedded tissue sections are used with specific anti-FABP9 antibodies. Unlike FABP6 which shows nuclear staining, FABP9 staining is observed exclusively in the cytoplasm of prostate cells . The staining intensity is generally classified into four categories:

• Unstained (0): No detectable staining

• Weakly stained (+): Faint cytoplasmic staining

• Moderately stained (++): Clear cytoplasmic staining

• Strongly stained (+++): Intense cytoplasmic staining

This semi-quantitative assessment enables correlation between FABP9 expression levels and clinical parameters such as Gleason score and androgen receptor index .

What are the appropriate control tissues when studying FABP9 expression?

When examining FABP9 expression in prostate cancer, benign prostatic hyperplasia (BPH) tissues serve as appropriate controls. Research has shown distinct expression patterns between BPH and carcinoma tissues:

As shown in this table, the majority of BPH cases (70%) show no FABP9 staining, while the remainder exhibit only weak staining. This contrasts significantly with carcinoma tissues, where all samples show at least weak FABP9 expression, with most exhibiting moderate to strong staining . This clear differential expression makes BPH an excellent control tissue for FABP9 studies.

What is the correlation between FABP9 expression and Gleason scores?

Research has established a significant correlation between FABP9 expression and Gleason scores (GS) in prostate carcinomas. When analyzed according to malignancy levels:

Statistical analysis reveals that FABP9 staining intensity in moderately malignant cases is significantly higher than in low malignant cases (p = 0.007). Similarly, FABP9 expression is significantly higher in cases with high GS than in those with low GS (p < 0.001) . This progressive increase in FABP9 expression with increasing Gleason scores suggests that FABP9 could be a marker for prostate cancer aggression and progression.

How should FABP9 staining intensity be quantified in immunohistochemistry?

Quantification of FABP9 staining in immunohistochemistry should follow a systematic approach to ensure reproducibility. Based on established research protocols, a four-tier scoring system is recommended:

• Unstained (0): No detectable staining

• Weakly stained (+): Faint but discernible cytoplasmic staining

• Moderately stained (++): Clear cytoplasmic staining of intermediate intensity

• Strongly stained (+++): Intense cytoplasmic staining

For statistical analysis, these categories can be used directly in non-parametric tests (such as Chi-square test for comparing frequencies between groups) or can be converted to numerical values (0, 1, 2, 3) for correlation analyses with clinical parameters . When analyzing correlation with clinical factors such as Gleason score or androgen receptor index, the Mann-Whitney U test has proven effective . It's important to note that FABP9 staining is exclusively cytoplasmic in prostate cancer cells, which differs from other FABP family members like FABP6 that show nuclear localization.

How can FABP9 expression be reliably quantified in cell line models?

Reliable quantification of FABP9 expression in cell line models requires a multi-level approach addressing both mRNA and protein expression:

For mRNA quantification, quantitative reverse transcription PCR (qRT-PCR) is the method of choice. Primers should be designed specifically for FABP9 to avoid cross-amplification with other FABP family members. Example primer sequences used in research include:

For protein quantification, Western blot analysis provides reliable results, with FABP9 typically appearing as a single band at approximately 14 kDa . When comparing cell lines, research has shown that FABP9 protein is highly expressed in highly malignant prostate cancer cell lines (PC-3 and PC3-M) but may be undetectable in benign PNT-2 cells . This differential expression makes these cell lines useful as positive and negative controls when establishing quantification methods.

What are the technical considerations for FABP9 knockdown experiments?

FABP9 knockdown experiments require careful technical considerations to ensure effective suppression. Based on published research approaches:

• siRNA design and selection:

  • Multiple siRNAs targeting different regions of FABP9 mRNA should be tested.

  • Published research has shown varying efficacy with different siRNAs, with the most effective achieving approximately 60% reduction in protein expression .

• Transfection optimization:

  • Cell density at transfection time significantly affects knockdown efficiency.

  • A transfection duration of 48 hours has been shown to be effective for FABP9 knockdown .

• Validation of knockdown:

  • Western blot analysis is essential to confirm protein reduction.

  • Include appropriate controls: untreated cells, scramble siRNA controls, and transfection reagent-only controls .

• Functional assays:

  • Research indicates that FABP9 knockdown affects invasive potential but may not impact growth rate, anchorage-independent growth, or migration rate .

  • Design experiments to assess these phenotypes separately to distinguish specific effects of FABP9 reduction.

What is the relationship between FABP9 expression and androgen receptor signaling?

Research has revealed a significant correlation between FABP9 expression and androgen receptor (AR) index in prostate cancer tissues. Box plot analyses have demonstrated that AR index levels are significantly higher in cases with strong FABP9 staining compared to those with weak FABP9 staining (Mann-Whitney U test, p = 0.03) .

When investigating this relationship, researchers should consider:

• Dual immunohistochemical staining approaches to simultaneously visualize FABP9 and AR in the same tissue sections.

• Correlation analyses between FABP9 staining intensity and AR index using appropriate statistical methods.

• Mechanistic studies to determine whether:

  • AR signaling regulates FABP9 expression

  • FABP9 modulates AR signaling

  • The two pathways are independently regulated but converge on common downstream targets

Understanding this relationship may provide insights into how FABP9 contributes to prostate cancer progression and potentially offer new therapeutic targets for intervention, particularly in advanced disease states.

How can recombinant FABP9 be generated for functional studies?

Generating high-quality recombinant FABP9 for functional studies involves several critical steps:

• Expression vector construction:

  • Clone the full-length FABP9 open reading frame into an expression vector.

  • Include an affinity tag for purification (His-tag is commonly used).

  • Suitable vectors include pET28a with NdeI and XhoI restriction sites .

• Expression system:

  • E. coli BL21-DE5 competent cells have been successfully used for FABP9 expression .

• Protein purification:

  • For His-tagged FABP9, use Ni-NTA agarose affinity chromatography.

  • Include appropriate washing steps to remove non-specifically bound proteins.

• Functional validation:

  • In vitro cellular adhesion assays can be used to test recombinant FABP9 functionality.

  • Coat Ni-NTA agarose beads with purified recombinant FABP9.

  • Incubate with appropriate cell types and observe cellular interactions via light microscopy .

This approach ensures production of functional recombinant FABP9 suitable for a range of experimental applications.

How do we interpret discrepancies between FABP9 mRNA and protein expression levels?

Discrepancies between FABP9 mRNA and protein levels require careful interpretation. In research studies, FABP9 mRNA levels were elevated in all malignant prostate cell lines compared to benign cells, but protein expression was only detectable in highly malignant PC-3 and PC3-M cells . This suggests context-dependent post-transcriptional regulation that might be biologically significant in cancer progression.

When facing such discrepancies, consider:

• Post-transcriptional regulation:

  • MicroRNAs may target FABP9 mRNA, reducing translation efficiency.

  • RNA-binding proteins might alter mRNA stability or translation.

• Protein stability differences:

  • Variations in FABP9 protein turnover rates across different cell types.

  • Consider pulse-chase experiments to assess FABP9 protein half-life.

• Technical considerations:

  • Verify qRT-PCR primer specificity for accurate mRNA quantification.

  • Validate antibody specificity for protein detection methods.

It's advisable to rely on protein-level measurements for functional interpretations while using mRNA data to understand transcriptional regulation.

What experimental designs best elucidate FABP9's role in cancer progression?

To comprehensively understand FABP9's role in cancer progression, multiple experimental approaches should be combined:

• Clinical correlation studies:

  • Analyze FABP9 expression in tissue microarrays with patient follow-up data.

  • Correlate expression with Gleason scores, androgen receptor index, and survival outcomes.

  • Conduct multivariate analyses to determine if FABP9 is an independent prognostic factor.

• In vitro functional studies:

  • Modulate FABP9 expression through siRNA-mediated knockdown or overexpression.

  • Assess impact on cancer-related phenotypes, particularly invasion assays, as research indicates FABP9 specifically affects invasion but not other phenotypes .

• Signaling pathway analyses:

  • Investigate FABP9's interaction with the androgen receptor pathway.

  • Explore connections to known invasion-related pathways.

• In vivo models:

  • Develop orthotopic prostate cancer models with modulated FABP9 expression.

  • Assess tumor growth, local invasion, and metastatic potential.

This comprehensive approach will provide insights into both the mechanistic role of FABP9 in cancer progression and its potential clinical utility as a biomarker.

How should FABP9 antibody specificity be validated?

Validating FABP9 antibody specificity is crucial for obtaining reliable research results. A comprehensive validation approach should include:

• Western blot analysis:

  • Test the antibody against recombinant FABP9 protein as a positive control.

  • Verify a single band at the expected molecular weight (approximately 14 kDa) .

  • Include negative controls such as cell lines known to lack FABP9 expression (e.g., benign PNT-2 prostate cells).

  • Test for cross-reactivity with other FABP family members.

• Immunohistochemical validation:

  • Verify cytoplasmic-only staining pattern in prostate carcinoma cells (distinguishing from FABP6, which shows nuclear staining) .

  • Include absorption controls by pre-incubating the antibody with recombinant FABP9 protein.

• Correlation with mRNA expression:

  • Verify that tissues or cell lines with high FABP9 mRNA levels also show protein expression when detected with the antibody.

• Knockdown validation:

  • Demonstrate proportional reduction in antibody signal in samples where FABP9 has been knocked down via siRNA .

These rigorous validation steps ensure that observed signals truly represent FABP9 expression and not artifacts or cross-reactivity with other proteins.

What approaches can be used to study the functional impact of FABP9 in cellular invasion?

To study FABP9's role in cellular invasion, several complementary approaches can be employed:

• RNAi-mediated knockdown:

  • Use siRNA to suppress FABP9 expression in highly invasive cell lines like PC3-M.

  • Published research shows that effective knockdown (up to 60% reduction) can be achieved with optimized siRNA .

  • Validate knockdown efficiency by Western blotting, comparing with scramble siRNA controls.

• Invasion assays:

  • Use Matrigel-coated transwell chambers to quantitatively assess invasive potential.

  • Compare invasion rates between FABP9-knockdown cells and control cells.

  • Research has demonstrated that FABP9 suppression inhibits invasive potential in highly malignant PC3-M cells .

• Specificity controls:

  • Test whether FABP9 knockdown affects other cancer-related phenotypes:

    • Proliferation rates (cell counting, MTT assays)

    • Anchorage-independent growth (soft agar colony formation)

    • Migration (wound healing assays)

  • Research indicates FABP9 specifically regulates invasion but not these other phenotypes .

• Mechanistic investigations:

  • Analyze expression of invasion-related genes (MMPs, integrins) following FABP9 modulation.

  • Investigate potential connections between FABP9 and known invasion pathways.

This multi-faceted approach will provide comprehensive insights into FABP9's specific role in cancer cell invasion.