SH3GLB1 Antibody

What is SH3GLB1 and what cellular functions does it regulate?

SH3GLB1 belongs to the endophilin family and contains an N-terminal BAR domain and a C-terminal SH3 domain implicated in various signaling pathways and membrane reshaping activities . The protein is involved in several critical cellular functions:

• Membrane curvature function: SH3GLB1 participates in mitochondrial dynamics, autophagy regulation, apoptotic processes, and endocytosis through its membrane reshaping capabilities .

• Autophagy initiation: SH3GLB1 is indispensable for the initiation of autophagy, recruiting beclin1 and activating PI3KC3, which are crucial steps for forming early autophagosomes .

• Mitochondrial homeostasis: The protein maintains mitochondrial function and influences mitochondrial metabolism .

• Neuroprotection: SH3GLB1 protects neuronal cells from amyloid-β-induced cytotoxicity, suggesting its importance in brain physiology .

How does SH3GLB1 expression correlate with disease outcomes in cancer?

Analysis of clinical databases and tumor samples has revealed significant correlations between SH3GLB1 expression and cancer outcomes:

• Disease grading: High SH3GLB1 gene expression is associated with higher disease grading in glioblastoma .

• Survival analysis: Both CGGA and TCGA databases demonstrate that patients with higher SH3GLB1 expression show worse prognosis and survival profiles .

• Tumor recurrence: SH3GLB1 expression is significantly elevated in recurrent tumors compared to primary ones, with protein expression enhanced in eight of nine recurrent tumor tissues examined in one study .

• Clinical correlation: There is a positive correlation between the levels of SH3GLB1 and CD133 (a tumor-initiating cell marker) in GBM according to clinical database analysis .

What are recommended methods for detecting SH3GLB1 in experimental settings?

For effective detection of SH3GLB1 in research applications, several validated methods can be employed:

• Western blotting: Use antibodies from validated sources (e.g., Proteintech) for protein level quantification . This technique is particularly useful for comparing SH3GLB1 levels between parental and resistant cell lines or between different cellular fractions.

• Immunofluorescence: This method reveals that SH3GLB1 is distributed in the nucleus, cytoplasm, and mitochondria, allowing for subcellular localization studies .

• RNA interference: siRNA targeting SH3GLB1 (specific sequences: 5′-GGGAAUCAGCAGUACACAUTT-3′ and 3′-AUGUGUACUGCUGAUUCCCTT-5′) can be used with Lipofectamine® RNAiMAX reagent for transient knockdown .

• Stable gene modulation: For long-term studies, lentiviral-transfected cells using SH3GLB1 shRNA or overexpression vectors can be selected to create stable cell lines .

What controls should be included when studying SH3GLB1 function?

When designing experiments to study SH3GLB1 function, include these essential controls:

• Housekeeping protein controls: Use actin or vinculin as loading controls for western blotting experiments .

• Empty vector controls: For overexpression studies, include cells transfected with empty vectors to control for transfection effects.

• Scrambled/non-targeting siRNA: When using RNA interference, include a non-targeting siRNA control to account for non-specific effects of the transfection process .

• Parental vs. resistant cell comparisons: When studying drug resistance mechanisms, compare parental cells with their derived resistant lines (e.g., U87MG vs. U87MG-R, A172 vs. A172-R) .

• Pathway inhibitor controls: Include autophagy inhibitors like chloroquine when studying SH3GLB1's role in autophagy-mediated processes .

How does SH3GLB1 mechanistically regulate CD133 expression in glioblastoma cells?

SH3GLB1 influences CD133 expression through epigenetic regulation of the CD133 promoter region:

• Nuclear localization: Immunofluorescence studies show that SH3GLB1 is distributed in the nucleus, enabling direct gene regulation functions .

• Histone acetylation modulation: SH3GLB1 regulates CD133 expression by influencing histone H4K5 acetylation. When SH3GLB1 is downregulated in resistant cells, both histone H4K5 acetylation and CD133 levels are reduced .

• Promoter binding: ChIP assays demonstrate that histone H4K5 acetylation occurs in the CD133 promoter region, with significantly enhanced acetylation levels in resistant cells compared to parental cells .

• Bidirectional regulation: SH3GLB1 overexpression in parental cells enhances H4K5 acetylation on the CD133 promoter, while SH3GLB1 knockdown in resistant cells reduces this acetylation .

• Promoter activity: Luciferase reporter assays confirm that CD133 promoter activity is enhanced with SH3GLB1 upregulation .

This epigenetic mechanism provides a molecular link between SH3GLB1 and the regulation of tumor-initiating cell features through CD133 expression.

What is the relationship between SH3GLB1, autophagy, and mitochondrial function in drug resistance?

SH3GLB1 orchestrates a complex interplay between autophagy and mitochondrial metabolism that contributes to drug resistance:

• Autophagy regulation: Downregulation of SH3GLB1 results in retention of TMZ susceptibility, upregulated p62, and reduced LC3B-II, indicating disrupted autophagy .

• OXPHOS modulation: Autophagy inhibition via SH3GLB1 deficiency and chloroquine treatment leads to attenuated oxidative phosphorylation (OXPHOS) expression .

• Mitochondrial metabolism: SH3GLB1 inhibition in resistant cells alleviates TMZ-enhanced mitochondrial metabolic functions, including:

  • Reduced mitochondrial membrane potential

  • Decreased mitochondrial respiration

  • Lower ATP production

• Pathway integration: Increased SH3GLB1 promotes autophagy, which enhances mitochondrial function through OXPHOS, ultimately contributing to TMZ resistance in glioblastoma cells .

• In vivo confirmation: Animal models demonstrate that resistant tumor cells with SH3GLB1 knockdown become resensitized to TMZ, with suppression of TMZ-induced autophagy and OXPHOS .

This mechanistic pathway suggests that targeting SH3GLB1 could disrupt the autophagy-mitochondrial axis that drives drug resistance.

How can single-cell RNA sequencing data inform our understanding of SH3GLB1's role in tumor heterogeneity?

Single-cell RNA sequencing analysis reveals important insights about SH3GLB1's role in tumor heterogeneity:

• Cluster identification: scRNA transcriptomic analysis of five GBM tumors identified distinct cell clusters based on gene expression differences, with five clusters (1, 2, 4, 5, and 6) identified as tumor cells .

• TIC marker correlation: Cluster 4 exhibited the most abundant expression of tumor-initiating cell markers including CD133, Olig2, SOX2, Bmi1, and Myc .

• SH3GLB1 expression pattern: Clusters 1 and 4 showed significantly higher expressions of SH3GLB1 compared to clusters 2 and 6, which had the lowest levels of TIC profiles .

• Functional implications: This pattern suggests that SH3GLB1 expression is enriched in tumor cell populations with stem-like characteristics, providing insight into intratumoral heterogeneity related to treatment resistance.

• Targeting strategy: The correlation between SH3GLB1 expression and TIC markers in specific cell populations suggests that targeting SH3GLB1 could potentially address the more aggressive subpopulations within heterogeneous tumors.

These findings demonstrate how single-cell analysis can reveal cell-type specific roles of SH3GLB1 that might be masked in bulk tissue analysis.

What experimental approaches can determine if SH3GLB1 mediates tumor-initiating cell features?

To investigate SH3GLB1's role in tumor-initiating cell characteristics, researchers can employ several sophisticated experimental approaches:

• Extreme Limiting Dilution Assay (ELDA): This in vitro technique demonstrated that stemness frequency decreased from 1/4.21 to 1/57.42 in U87MG-R cells and from 1/3.23 to 1/11.26 in A172-R cells following SH3GLB1 knockdown .

• Spheroid formation assays: Loss of SH3GLB1 in primary TMZ-resistant cells disrupted tumor sphere formation, indicating impaired self-renewal capacity .

• CD133+ cell isolation and viability testing: CD133+ cells from resistant cells with enhanced SH3GLB1 levels were shown to more easily survive cytotoxic treatment than those from parental cells .

• Co-culture experiments: Different percentages of resistant CD133+ cells co-cultured with parental cells revealed that resistant CD133+ cells with high SH3GLB1 levels contributed to the main survivors after TMZ treatment .

• Marker expression analysis: Western blotting to examine changes in TIC markers (like CD133) following SH3GLB1 modulation provides mechanistic insights into how this protein affects stemness characteristics .

These functional assays collectively establish SH3GLB1's critical role in maintaining tumor-initiating cell phenotypes that contribute to treatment resistance.

What are the technical considerations for ChIP assays when investigating SH3GLB1's effect on CD133 promoter acetylation?

When using Chromatin Immunoprecipitation (ChIP) assays to study SH3GLB1's impact on CD133 promoter acetylation, researchers should consider these technical aspects:

• Antibody selection: Use highly specific antibodies against histone H4K5 acetylation for immunoprecipitation to ensure accurate detection of the modification .

• Cell model considerations: Compare results between parental and resistant cell lines (U87MG vs. U87MG-R, A172 vs. A172-R) to understand context-dependent effects .

• Primary cell validation: Confirm findings from cell lines in primary clinical cells to ensure physiological relevance .

• Promoter region targeting: Design primers that specifically amplify the CD133 promoter region containing H4K5 acetylation sites .

• Controls: Include input controls (non-immunoprecipitated chromatin) and IgG controls (non-specific antibody) to account for background binding.

• Complementary approaches: Validate ChIP findings with luciferase reporter assays to confirm functional relevance of the identified acetylation changes .

• SH3GLB1 modulation: Perform ChIP assays under conditions of SH3GLB1 overexpression and knockdown to establish causality between SH3GLB1 levels and H4K5 acetylation .

These considerations ensure robust and reproducible ChIP data when investigating the epigenetic mechanisms of SH3GLB1-mediated CD133 regulation.

How might targeting SH3GLB1 be incorporated into combination therapy strategies for glioblastoma?

Based on the mechanistic understanding of SH3GLB1's functions, several rational combination therapy approaches could be explored:

• Autophagy modulation: Combining SH3GLB1 inhibition with autophagy inhibitors like chloroquine could synergistically suppress the autophagy-dependent resistance mechanisms in GBM .

• Epigenetic therapy: Since SH3GLB1 regulates CD133 expression through histone H4K5 acetylation, combining SH3GLB1 targeting with histone deacetylase inhibitors could potentially enhance efficacy against tumor-initiating cells .

• Metabolic targeting: SH3GLB1 knockdown attenuates oxidative phosphorylation in resistant cells, suggesting that combining SH3GLB1 inhibition with OXPHOS inhibitors could more effectively disrupt cancer cell metabolism .

• Standard-of-care integration: In animal models, SH3GLB1 knockdown resensitized resistant tumor cells to TMZ, indicating that SH3GLB1 inhibition could be effectively combined with standard chemotherapy for enhanced efficacy .

• TIC-targeting strategies: Given SH3GLB1's role in maintaining tumor-initiating cell features, combining SH3GLB1 inhibition with other stem cell-targeting approaches could address the resistant subpopulations responsible for tumor recurrence .

These combination strategies leverage the multiple cellular processes regulated by SH3GLB1 to potentially overcome the multifaceted resistance mechanisms in glioblastoma.