ABCI1 Antibody

What is ACBI1 and what is its mechanism of action in research applications?

ACBI1 functions as a dual degrader specifically targeting the SMARCA2/4 ATPases of the SWI/SNF chromatin remodeling complex. The compound effectively inhibits neuroblastoma (NB) cell proliferation, invasion, and notably, cellular plasticity by disrupting chromatin accessibility . Mechanistically, ACBI1 leads to the degradation of SWI/SNF ATPases, resulting in chromatin compaction at specific genomic loci. This compaction reduces the binding of core transcription factors and affects histone modifications, particularly H3K27ac, which marks active enhancers and promoters . When designing experiments with ACBI1, researchers should consider time-dependent effects, as changes in chromatin structure precede alterations in gene expression profiles and cellular morphology.

How does ACBI1 treatment affect chromatin structure and transcription factor binding?

ACBI1 treatment significantly alters chromatin structure, primarily by reducing chromatin accessibility at specific genomic sites. ChIP-seq analyses reveal that ACBI1 treatment causes decreased binding of core transcription factors such as HAND2 and PHOX2B at compacted sites in neuroblastoma cells . Additional ChIP-seq experiments demonstrated reduced signals for the cohesin subunit RAD21, transcription repressor CTCF, and histone-methylating complex subunit WDR5 at these compacted sites .

Interestingly, the effect on transcription factor binding is complex and target-specific. While ACBI1 decreases HAND2 and PHOX2B protein levels by approximately 30% and reduces H3K27ac levels by 15%, it paradoxically increases MYCN and GATA3 ChIP-seq signals at sites with unchanged chromatin accessibility . This suggests a potential redistribution of certain transcription factors from compacted sites to regions that maintain open chromatin structure.

What controls should be included when studying ACBI1 effects on gene expression?

When designing experiments to study ACBI1's effects on gene expression, researchers should implement several crucial controls. First, include vehicle controls (DMSO) to account for solvent effects. Second, perform time-course experiments to capture both immediate and delayed transcriptional responses. Third, include protein level measurements (Western blot) alongside transcriptomic analyses to distinguish between direct effects on transcription versus indirect effects through protein stability.

Based on published data, Western blot assays should monitor PHOX2B, HAND2, H3K27ac, MYCN, and GATA3 levels as these show differential responses to ACBI1 treatment . Additionally, chromatin accessibility assays (ATAC-seq) should be performed in parallel with transcriptomic analyses to correlate changes in gene expression with alterations in chromatin structure.

How can ACBI1 be used to study cellular plasticity in neuroblastoma models?

ACBI1 represents a powerful tool for investigating cellular plasticity in neuroblastoma models due to its ability to impair the transition between different cell states. To effectively use ACBI1 for studying cell plasticity, researchers should employ a methodological approach that combines morphological assessment with molecular profiling.

A recommended experimental design involves culturing neuroblastoma cells (such as SJNBL012407_X1) in stem cell media (SCM) followed by transfer to FBS-containing media to induce heterogeneous cell populations . Apply ACBI1 treatment (typically 250nM) either before or during this transition. Monitor morphological changes using time-lapse microscopy with the Incucyte system to quantify cell type transitions. For molecular characterization, perform both bulk RNA-seq and single-cell RNA-seq (scRNA-seq) to capture population-level and cell-specific transcriptional changes .

What is the relationship between ACBI1 treatment and neuronal differentiation pathways?

ACBI1 treatment shows a complex relationship with neuronal differentiation pathways in neuroblastoma, making it a valuable tool for studying neural development mechanisms. Transcriptomic analyses reveal that ACBI1 promotes expression of genes associated with neuronal differentiation while simultaneously suppressing adrenergic and mesenchymal programs .

To study this phenomenon, researchers should implement a multi-level analytical approach. Begin with bulk RNA-seq to identify broad transcriptional changes, focusing on gene sets associated with immature dopaminergic neurons (HDA) and outer radial glial cells undergoing neuronal differentiation, which show positive enrichment following ACBI1 treatment . Follow with scRNA-seq to characterize heterogeneity in cellular responses, as ACBI1 treatment increases the population of cells exhibiting high HDA signatures compared to vehicle-treated controls .

For functional validation, combine ACBI1 treatment with established neuronal differentiation protocols using retinoic acid or other differentiation agents. Monitor neuronal marker expression using immunofluorescence or flow cytometry targeting dopaminergic markers. Additionally, evaluate morphological changes associated with neuronal differentiation, such as neurite outgrowth and decreased proliferation rates.

What are optimal approaches for combining ACBI1 with other therapeutic agents in research models?

When investigating ACBI1 in combination with other therapeutic agents, researchers should employ systematic approaches to properly characterize potential synergistic effects. Based on published data, ACBI1 shows promising synergy with chemotherapeutic agents like etoposide in neuroblastoma models .

A comprehensive methodology for combination studies should include:

• Dose-response matrix experiments: Test a range of concentrations for both ACBI1 and the combination agent to generate complete dose-response surfaces.

• Synergy quantification: Calculate Bliss synergy scores across the dose range. Significant synergy is indicated by scores greater than 10, as observed with ACBI1-etoposide combinations .

• Temporal considerations: Evaluate sequential versus simultaneous administration of compounds.

• Multiparametric assessment: Include cell confluence assays, viability measurements (CellTiter-Glo), and morphological analysis to capture the full spectrum of cellular responses .

When specifically combining ACBI1 with etoposide, optimal experimental conditions include 250nM ACBI1 and 500nM etoposide, as this combination demonstrates greater efficacy than either agent alone . For morphological analysis, use high-content imaging to distinguish between different cellular phenotypes - ACBI1 treatment typically yields spheres and neuroblast-like cells, while etoposide reduces adrenergic-type cell numbers but leaves flattened monolayer cells largely intact .

How does ACBI1 treatment affect cell cycle progression and cytotoxicity in different cellular contexts?

ACBI1 exhibits context-dependent effects on cell cycle progression and cytotoxicity that vary by cell type and culture conditions. To properly investigate these effects, researchers should implement a comprehensive experimental design that distinguishes between cytostatic and cytotoxic responses.

For cytotoxicity assessment, the Incucyte Cytotox Green Dye system provides valuable real-time data on cell death. Published data indicates that ACBI1 treatment induces modest cell death (<10%) in neuroblastoma cultures, affecting both adrenergic-like large spheres and cells outside these spheres . To differentiate between primary and secondary effects, perform time-course experiments measuring cell death at multiple timepoints (24h, 48h, 72h) after ACBI1 administration.

Cell cycle analysis should combine flow cytometry for population-level assessments with time-lapse microscopy for single-cell tracking. This approach reveals that ACBI1's primary effect is often growth inhibition rather than direct cytotoxicity. When analyzing heterogeneous cultures, such as PDX-derived neuroblastoma cells transitioning from stem cell media to FBS-containing conditions, ACBI1 significantly decreases cell proliferation while inducing limited cell death .

For mechanistic studies, combine these functional assays with molecular analyses targeting cell cycle regulators and apoptotic pathways. The strong growth-inhibitory effect of ACBI1, particularly in combination with its ability to alter cell plasticity, suggests its primary mechanism involves disrupting the balance between proliferation and differentiation programs rather than direct cytotoxicity.

What are the best practices for analyzing ChIP-seq data following ACBI1 treatment?

Analyzing ChIP-seq data after ACBI1 treatment requires specific considerations due to the compound's effects on chromatin accessibility and transcription factor binding. The methodological approach should account for both global and site-specific changes in protein-DNA interactions.

Given that ACBI1 distinctly affects chromatin accessibility, integrate ATAC-seq data with ChIP-seq analysis to classify genomic regions as either "compacted" or "unchanged" following treatment . This classification is critical for properly interpreting changes in transcription factor binding. Specifically, analyze factor binding at both compacted sites and sites with unchanged accessibility separately, as demonstrated in published work where HAND2, PHOX2B, and H3K27ac showed decreased binding at both site types, while MYCN and GATA3 exhibited increased binding at sites with stable accessibility .

For metagene analysis, center plots on transcription start sites or enhancer regions and extend the analysis window to at least ±2kb to capture broader chromatin environment changes. Use appropriate normalization methods - spike-in normalization is recommended when expecting global changes in factor binding. Finally, correlate ChIP-seq findings with protein level changes measured by Western blot to distinguish between effects on protein abundance versus chromatin binding affinity.

How should researchers design experiments to investigate ACBI1 effects on cellular heterogeneity and differentiation?

Investigating ACBI1's effects on cellular heterogeneity and differentiation requires a multifaceted experimental design that captures changes at both population and single-cell levels. Based on published methodologies, researchers should implement the following approach:

First, establish baseline cellular heterogeneity by culturing cells under conditions that promote diverse phenotypes. For neuroblastoma research, this typically involves transitioning cells from stem cell media to FBS-containing media for 3-5 days to generate heterogeneous populations . Document initial heterogeneity through morphological assessment and expression profiling of lineage markers.

For intervention studies, apply ACBI1 at established concentrations (typically 250nM) either before or during the transition to heterogeneity-inducing conditions. Include appropriate controls, particularly DMSO-treated cells cultured under identical conditions. Monitor morphological changes through high-resolution time-lapse imaging, focusing on the emergence or suppression of distinct cellular morphologies.

Finally, validate findings through functional assays specific to the differentiation pathways of interest, combining the power of population-based assays with single-cell resolution techniques.

What are the key considerations for validating ACBI1 specificity in experimental systems?

Validating ACBI1 specificity requires a systematic approach focusing on both on-target engagement and off-target effects. The following methodological framework ensures comprehensive validation:

First, confirm target engagement through protein degradation assays. Western blotting should demonstrate specific reduction of SMARCA2/4 protein levels without affecting other SWI/SNF complex components. Time-course experiments are essential to establish the kinetics of target degradation, typically examining protein levels at 4, 8, 24, and 48 hours post-treatment.

For mechanistic validation, perform rescue experiments using degradation-resistant SMARCA2/4 mutants. If ACBI1 effects stem specifically from SMARCA2/4 degradation, expressing these mutants should attenuate the observed phenotypes. Additionally, compare ACBI1 effects with genetic knockdown approaches (siRNA or CRISPR) targeting SMARCA2/4 to confirm phenotypic concordance.

To assess off-target effects, conduct proteomics analysis using techniques like tandem mass tag (TMT) labeling to identify proteins whose abundance changes following ACBI1 treatment. Gene expression profiling should compare ACBI1 treatment with selective knockdown of SMARCA2/4 to distinguish on-target transcriptional changes from potential off-target effects.

Finally, dose-response studies are critical for establishing the concentration range where ACBI1 maintains target specificity. Correlate the degree of SMARCA2/4 degradation with functional phenotypes across a concentration range to identify the optimal working concentration that maximizes on-target effects while minimizing potential off-target activities.

How should researchers analyze transcriptomic data following ACBI1 treatment to identify key regulatory pathways?

Analyzing transcriptomic data following ACBI1 treatment requires a structured analytical approach that identifies both direct and indirect effects on gene expression. Based on published methodologies, researchers should implement the following comprehensive workflow:

Begin with standard differential expression analysis comparing ACBI1-treated samples to vehicle controls. Apply appropriate statistical thresholds (typically adjusted p-value <0.05 and fold change >1.5) while considering the biological context - ACBI1 effects may be more subtle than classical transcriptional inhibitors. For neuroblastoma studies, research has shown that ACBI1 treatment results in negative enrichment of adrenergic (ADRN), mesenchymal (MES), EMT, and collagen-containing extracellular matrix (CCEM) signature genes .

Next, perform pathway enrichment analysis using established databases (KEGG, GO, MSigDB) to identify affected biological processes. Given ACBI1's mechanism as a SWI/SNF ATPase degrader, focus particular attention on chromatin organization, transcriptional regulation, and cell differentiation pathways. Additionally, conduct transcription factor enrichment analysis to identify key regulators whose activity is affected by chromatin remodeling changes.

Integrate ATAC-seq data with transcriptomic results to correlate changes in gene expression with alterations in chromatin accessibility. This approach helps distinguish genes directly affected by chromatin compaction from those responding secondarily. Research has shown positive enrichment of genes associated with immature dopaminergic neurons and neuronal differentiation following ACBI1 treatment , suggesting activation of specific developmental programs.

Finally, implement time-course analysis to distinguish immediate from delayed transcriptional responses, providing insight into the sequential unfolding of regulatory events following SWI/SNF ATPase degradation.

What are promising research applications for ACBI1 beyond neuroblastoma models?

ACBI1's mechanism as a dual degrader of SMARCA2/4 opens numerous research applications beyond neuroblastoma. The compound's demonstrated ability to modulate chromatin accessibility, transcription factor binding, and cellular plasticity makes it valuable for investigating fundamental aspects of chromatin biology across multiple experimental systems.

In cancer research, ACBI1 could be applied to other malignancies where SWI/SNF dysregulation plays a role, such as lung cancer, prostate cancer, and bladder cancer. Current research indicates ABCC1 overexpression predicts unfavorable outcomes in bladder urothelial carcinoma, kidney papillary cell carcinoma, brain lower grade glioma, hepatocellular carcinoma, and several other cancers . Investigating how ACBI1 affects cellular plasticity and differentiation in these contexts could provide insights into resistance mechanisms and potential therapeutic combinations.

For developmental biology, ACBI1 represents a valuable tool for studying the role of chromatin remodeling in lineage specification and cellular differentiation. The compound's ability to promote neuronal differentiation pathways suggests applications in neural development research, potentially extending to induced pluripotent stem cell (iPSC) differentiation protocols.

In the field of transcriptional regulation, ACBI1 could serve as a probe for investigating enhancer-promoter interactions and the dynamics of transcription factor binding site selection. The differential effects on transcription factor binding (decreased for some factors, increased for others) raise interesting questions about the principles governing factor recruitment to chromatin.

How might ACBI1 be used to investigate the relationship between chromatin accessibility and immune cell recruitment in cancer models?

ACBI1 presents a unique opportunity to investigate the complex relationship between chromatin remodeling and anti-tumor immunity. Given that ABCC1 expression has been shown to correlate with immune cell infiltration across multiple cancer types , ACBI1's ability to modulate SWI/SNF activity could provide mechanistic insights into this relationship.

A comprehensive experimental approach should begin with in vitro co-culture systems. Researchers can treat cancer cells (such as neuroblastoma or hepatocellular carcinoma lines) with ACBI1, then co-culture them with immune cells (macrophages, T cells, NK cells) to assess changes in immune cell activation, cytokine production, and cancer cell recognition. Published research indicates that ABCC1 expression in hepatocellular carcinoma cells positively correlates with macrophage infiltration , suggesting ACBI1 treatment might alter this relationship.

For in vivo studies, implement syngeneic mouse models with intact immune systems to evaluate how ACBI1 treatment affects tumor immune microenvironment. Use multi-parameter flow cytometry and spatial transcriptomics to characterize changes in immune cell composition, activation state, and spatial organization following treatment.

At the molecular level, integrate ATAC-seq, ChIP-seq, and RNA-seq analyses to identify ACBI1-induced changes in chromatin accessibility and expression of genes involved in immune signaling. Focus particularly on cytokine/chemokine genes, antigen presentation machinery, and immunomodulatory factors known to influence immune cell recruitment and activation.