SLFN11 Antibody

What is the molecular structure of SLFN11 protein?

SLFN11 has a molecular weight of approximately 102.8 kilodaltons . Its structure features a conserved core domain essential for function, while the N-terminal region exhibits significant diversity among Schlafen family members . The protein contains an ATPase domain that is crucial for its ability to promote stalled fork degradation in cells with DNA damage . This structural arrangement suggests a complex regulatory mechanism that may impact T cell development and differentiation, with implications for immune regulation and response to cellular stress .

How does SLFN11 affect cellular response to DNA-damaging agents?

SLFN11 sensitizes cells to a broad range of anti-cancer drugs including platinum derivatives (cisplatin and carboplatin), inhibitors of topoisomerases (irinotecan, topotecan, doxorubicin, etc.), DNA synthesis inhibitors (gemcitabine, cytarabine, hydroxyurea), and PARP inhibitors (olaparib, rucaparib, niraparib, talazoparib) . Despite their different mechanisms of action, all these drugs damage DNA during S-phase, activate the intra-S-phase checkpoint, and induce replication fork slowing and stalling. SLFN11 responds to this replication stress by irreversibly blocking replication, which explains the enhanced cytotoxicity in SLFN11-positive cells . Notably, SLFN11 expression is not correlated with the activity of protein kinase or tubulin inhibitors, confirming its specific role in the response to replication stress-inducing agents .

What techniques can be used to detect SLFN11 expression in research samples?

Several robust techniques are available for SLFN11 detection. Western blotting (WB) allows for quantitative analysis of protein expression levels. Immunoprecipitation (IP) enables the isolation and enrichment of SLFN11 from complex protein mixtures. Immunofluorescence (IF) visualizes SLFN11 cellular localization and distribution patterns. Enzyme-linked immunosorbent assay (ELISA) provides quantitative measurement of SLFN11 levels in various sample types . Additionally, researchers have developed and validated clinically applicable SLFN11 immunohistochemistry assays that can be used to assess SLFN11 expression in patient tumor samples . For visualization of SLFN11 foci, cells can be pre-extracted with 0.5% Triton X-100/CSK buffer, fixed with 4% paraformaldehyde, blocked with 2% BSA/PBS, and then stained with primary antibody .

How should researchers select the appropriate SLFN11 antibody for specific applications?

Selection of the appropriate SLFN11 antibody depends on the intended application and experimental design. For Western blotting and immunoprecipitation, researchers should consider mouse monoclonal antibodies like the IgG2a kappa light chain antibody that specifically detects human SLFN11 . For immunofluorescence studies, conjugated antibodies with fluorophores such as phycoerythrin (PE), fluorescein isothiocyanate (FITC), or Alexa Fluor® conjugates may be more suitable for direct visualization . For quantification of SLFN11 foci, FLAG-tagged constructs can be used with appropriate anti-FLAG antibodies . Researchers should validate antibody specificity for their particular application and cell/tissue type, and consider whether non-conjugated or conjugated antibodies are more appropriate for their specific experimental setup .

What are the recommended protocols for SLFN11 immunohistochemistry?

For immunohistochemistry (IHC) of SLFN11, clinically validated protocols have been developed . The general approach involves tissue fixation, antigen retrieval, blocking of endogenous peroxidase activity, and incubation with primary SLFN11 antibody. For quantification of SLFN11-FLAG foci in immunofluorescence studies, cells should be pre-extracted with 0.5% Triton X-100/CSK buffer (100 mM NaCl, 300 mM sucrose, 3 mM MgCl₂, 10 mM piperazine-1,4-bis[2-ethanesulfonic acid]) for 1 minute on ice, followed by fixation with 4% paraformaldehyde for 30 minutes at room temperature . After blocking with 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS), cells should be stained with primary antibody diluted in 2% BSA/PBS for 1 hour at room temperature . Quantification of foci can be performed using specialized software such as IN Cell Investigator 1.6 .

What is the mechanism of SLFN11-mediated stalled fork degradation?

SLFN11 promotes the degradation of stalled replication forks, a function that may contribute to the attrition of hematopoietic stem cells in Fanconi anemia (FA) . Research has revealed that loss of SLFN11 in FA cells partially restores tolerance to interstrand crosslinks (ICLs), associated with less pronounced mitomycin C (MMC)-induced chromosome breakage and reduced CHK1 phosphorylation and G2 phase arrest . The ATPase domain of SLFN11 is essential for promoting stalled fork degradation, as it affects RAD51 recruitment to reversed forks . This mechanism explains how SLFN11 expression influences cellular sensitivity to agents causing replication stress, as the degradation of stalled forks in SLFN11-positive cells leads to more severe DNA damage and subsequent cell death .

How does SLFN11 expression correlate with cancer treatment response?

SLFN11 expression strongly correlates with sensitivity to multiple classes of DNA-damaging agents and PARP inhibitors in cancer cells . Clinical evidence supports SLFN11 as a DNA-damaging agent (DDA) therapy selection biomarker in small cell lung cancer (SCLC) . Research has shown a trend for improved progression-free survival (PFS) in patients with SLFN11-positive tumors receiving standard of care therapies and olaparib maintenance, although this finding is caveated by small sample size . SLFN11 has been identified as a highly prevalent biomarker for PARP inhibitor response, independent of BRCA1/2 mutation status . Importantly, SLFN11 is inactivated in approximately 50% of cancer cell lines and a large fraction of tumors, highlighting its potential utility as a predictive biomarker for treatment response and resistance .

What is the relationship between SLFN11 and PARP inhibitor sensitivity?

SLFN11 expression has been identified as a causal determinant of response to PARP inhibitors in multiple studies . While BRCA-deficiency is traditionally associated with PARP inhibitor sensitivity, SLFN11 status has been shown to correlate with response independently of BRCA status or homologous recombination deficiency (HRD) scores . In the NCI-60 cell line panel, cells that do not express SLFN11 were found to be highly resistant to the PARP inhibitor talazoparib, and this correlation was validated in xenograft models . Studies in small cell lung cancer (SCLC) have identified SLFN11, but not homologous recombination genes or HRD scores, as a consistent determinant of response to PARP inhibitors . This suggests that SLFN11 expression status could serve as an additional biomarker for identifying patients likely to respond to PARP inhibitor therapy, beyond the current focus on BRCA mutations .

How might SLFN11 status inform combination therapy strategies?

The relationship between SLFN11 expression and drug sensitivity suggests several strategic approaches for combination therapies. For SLFN11-negative tumors, which are typically resistant to DNA-damaging agents and PARP inhibitors, combination with cell-cycle checkpoint inhibitors might overcome this resistance . Additionally, since SLFN11 is often silenced epigenetically, combination of DNA-targeting drugs with epigenetic modulators such as inhibitors of histone deacetylases (HDAC), DNA methyltransferases (DNMT), or EZH2 histone methyltransferase could potentially reactivate SLFN11 expression and restore sensitivity to these agents . These combination approaches represent promising strategies for addressing the approximately 50% of cancers that show low or absent SLFN11 expression and may otherwise be resistant to conventional DNA-damaging therapies .

What are the latest methodologies for measuring SLFN11 as a biomarker in clinical samples?

Recent advances in SLFN11 detection have led to the development and validation of clinically applicable immunohistochemistry assays . These assays allow for the assessment of SLFN11 tumor levels in patient samples and correlation with clinical outcomes . For research purposes, multiple techniques can be employed including western blotting, immunoprecipitation, immunofluorescence, and ELISA . Quantification of SLFN11 foci can be performed using specialized software such as IN Cell Investigator 1.6 . When developing biomarker assays for clinical use, researchers should focus on standardization and validation of the assay, including determination of appropriate cutoff values for SLFN11 positivity that correlate with clinical outcomes . The specific protocol and antibody selection should be guided by the intended application and the nature of the clinical samples being analyzed .

What epigenetic mechanisms regulate SLFN11 expression in cancer cells?

SLFN11 expression is frequently silenced in cancer through epigenetic mechanisms, making it a potential target for epigenetic therapy . Research suggests that inhibitors of histone deacetylases (HDAC), DNA methyltransferases (DNMT), and EZH2 histone methyltransferase can reactivate SLFN11 expression in cells where it has been epigenetically silenced . This reactivation could potentially restore sensitivity to DNA-damaging agents and PARP inhibitors in otherwise resistant tumors . Understanding the specific epigenetic modifications that regulate SLFN11 expression in different cancer types is an active area of research, with implications for developing targeted approaches to overcome treatment resistance . The development of methodologies to assess the epigenetic status of the SLFN11 gene in patient samples could provide additional biomarker information to guide treatment decisions .

What controls should be included when using SLFN11 antibodies in experiments?

When using SLFN11 antibodies, researchers should include appropriate positive and negative controls to ensure reliable results. Positive controls should include cell lines known to express high levels of SLFN11, while negative controls might include SLFN11-knockout cells generated using CRISPR/Cas9 or cells with siRNA-mediated SLFN11 silencing . For immunohistochemistry, tissue samples with known SLFN11 expression patterns should be used as controls . When optimizing new protocols, a titration of antibody concentrations should be performed to determine the optimal concentration for specific detection while minimizing background . For immunofluorescence studies involving quantification of foci, appropriate controls for the image analysis software should be established to ensure consistent and objective quantification .

How can researchers investigate the functional relationship between SLFN11 and DNA damage repair pathways?

To investigate the functional relationship between SLFN11 and DNA damage repair pathways, researchers can employ several methodologies. Comet assays, γH2AX foci formation, and chromosomal aberration analyses can assess DNA damage levels in SLFN11-positive versus SLFN11-negative cells exposed to various DNA-damaging agents . DNA fiber assays can evaluate replication fork progression and stability, which is particularly relevant given SLFN11's role in promoting stalled fork degradation . Co-immunoprecipitation and proximity ligation assays can identify protein-protein interactions between SLFN11 and components of DNA repair pathways. Cell cycle analysis using flow cytometry can determine how SLFN11 affects cell cycle progression in response to DNA damage . Finally, genetic approaches such as CRISPR/Cas9-mediated knockout or siRNA knockdown of SLFN11 in combination with various DNA repair factors can reveal synthetic lethal interactions and pathway dependencies .