UTP11 Antibody, HRP conjugated

What is UTP11 and why is it significant in cancer research?

UTP11 (also known as UTP11L) is a protein involved in nucleolar processing of pre-18S ribosomal RNA. Recent studies have revealed that UTP11 plays an important role in cancer development through multiple mechanisms:

• UTP11 is essential for the biosynthesis of 18S ribosomal RNAs by binding to the pre-rRNA processing factor MPP10

• It is frequently overexpressed in human cancers and associated with poor prognoses

• UTP11 deficiency inhibits cancer cell growth both in vitro and in vivo through p53-dependent and p53-independent mechanisms

• Depletion of UTP11 impedes 18S rRNA biosynthesis, triggering nucleolar stress

• It also represses SLC7A11 expression by promoting the decay of NRF2 mRNA, leading to enhanced ferroptosis

These findings position UTP11 as a potential oncogenic protein critical for cancer cell survival and a promising target for cancer research.

What is the function of HRP conjugation in antibodies used for UTP11 detection?

Horseradish peroxidase (HRP) conjugation serves as an enzymatic reporter system that enables highly sensitive detection of UTP11 protein. When conjugated to antibodies:

• The HRP enzyme catalyzes the oxidation of substrates in the presence of hydrogen peroxide, producing a detectable signal

• In chemiluminescent detection, HRP oxidizes luminol-based substrates, producing light that can be captured on film or by digital imaging systems

• This enzymatic amplification significantly enhances sensitivity compared to direct labeling methods, allowing detection of low-abundance proteins like UTP11 in cancer research

• The covalent linkage between antibody and HRP provides a stable conjugate that maintains both antibody specificity and enzymatic activity

This conjugation strategy is particularly valuable when studying proteins like UTP11 whose expression levels may vary significantly between normal and cancerous tissues.

What applications are most suitable for UTP11 HRP-conjugated antibodies?

UTP11 HRP-conjugated antibodies are versatile tools applicable across multiple research techniques:

• Western blotting: Enables quantitative analysis of UTP11 expression in cancer cell lines and tissue lysates, with high sensitivity for detecting both overexpression and knockdown phenotypes

• ELISA: Provides quantitative measurement of UTP11 in serum or cell lysates, useful for large-scale screening studies

• Immunohistochemistry (IHC): Allows visualization of UTP11 localization and expression patterns in tissue sections, particularly valuable for analyzing cancer biopsies

• Immunoprecipitation: When coupled with detection of binding partners like MPP10, helps elucidate UTP11's role in ribosome biogenesis

For nucleolar proteins like UTP11, immunocytochemistry with HRP-conjugated antibodies can also reveal subcellular localization patterns, confirming its nucleolar distribution and potential redistribution under stress conditions.

What advantages do direct HRP-conjugated UTP11 antibodies offer over two-step detection systems?

Direct HRP-conjugated primary antibodies provide several methodological advantages:

• Reduced protocol time: Elimination of secondary antibody incubation and associated wash steps shortens experimental time by several hours

• Increased specificity: Avoids potential cross-reactivity issues that can occur with secondary antibodies, particularly important when performing multi-protein analysis in complex cancer tissues

• Consistent labeling ratio: Methods like site-directed conjugation ensure uniform labeling of 1-2 HRP molecules per antibody, providing reproducible signal generation

• Simplified multiplexing: Enables simultaneous detection of multiple proteins without species cross-reactivity concerns

• Lower background: Elimination of secondary antibody binding to endogenous immunoglobulins reduces non-specific background

These advantages make direct HRP-conjugated UTP11 antibodies particularly valuable for high-throughput cancer screening applications and complex co-localization studies.

How can I optimize UTP11 antibody HRP conjugates for detecting low expression levels in normal tissues?

Detecting low UTP11 expression requires methodological optimization:

• Signal amplification systems:

  • Use enhanced chemiluminescent substrates specifically designed for low-abundance proteins

  • Consider tyramide signal amplification (TSA) which can increase sensitivity by 10-100 fold while maintaining specificity

• Sample preparation:

  • Enrich for nucleolar fractions where UTP11 is concentrated

  • Use phosphatase inhibitors during extraction as post-translational modifications may affect antibody recognition

• Antibody concentration optimization:

  • Perform titration experiments (1:500 to 1:5000) to determine optimal signal-to-noise ratio

  • Extended primary antibody incubation (overnight at 4°C) can improve detection of low-abundance targets

• Background reduction strategies:

  • Include 0.1-0.3% Triton X-100 in blocking solutions to reduce non-specific membrane interactions

  • Use milk-free blocking solutions as milk proteins can interfere with phospho-protein detection

  • Consider hydrogen peroxide pre-treatment to quench endogenous peroxidase activity

When analyzing normal tissues adjacent to tumors, these optimizations are crucial for establishing baseline UTP11 expression levels.

What is the molecular mechanism by which UTP11 deficiency triggers nucleolar stress, and how can HRP-conjugated antibodies help study this process?

UTP11 deficiency triggers nucleolar stress through a complex molecular cascade that can be investigated using HRP-conjugated antibodies:

• Ribosomal RNA processing disruption:

  • UTP11 binds to the pre-rRNA processing factor MPP10

  • Its depletion impedes 18S rRNA biosynthesis, disrupting ribosome assembly

  • HRP-conjugated antibodies against UTP11 and MPP10 can be used in co-immunoprecipitation studies to characterize this interaction

• Ribosomal protein redistribution:

  • Nucleolar stress causes ribosomal proteins RPL5 and RPL11 to bind to MDM2

  • This prevents MDM2-mediated p53 ubiquitination and degradation

  • Multiplexed immunoblotting with HRP-conjugated antibodies can track these protein interactions

• p53 stabilization and activation:

  • Activated p53 induces expression of growth arrest genes

  • HRP-conjugated antibodies against p53 and p21 can monitor this activation

  • Co-staining experiments can correlate UTP11 depletion with p53 accumulation

• NRF2-SLC7A11 axis disruption:

  • UTP11 deficiency promotes NRF2 mRNA decay

  • This reduces SLC7A11 expression, depleting glutathione and enhancing ferroptosis

  • RNA immunoprecipitation (RIP) followed by qPCR can analyze UTP11-mRNA interactions

These mechanistic studies benefit from the high sensitivity and specificity of HRP-conjugated antibodies, particularly in time-course experiments tracking the progression of nucleolar stress.

How can I validate the specificity of UTP11 HRP-conjugated antibodies in my experimental system?

Rigorous validation of UTP11 HRP-conjugated antibodies should include:

• Genetic validation approaches:

  • siRNA/shRNA knockdown: Compare signal in cells expressing UTP11 siRNA (5′-GAAGCTAAGAAAATCGAAA-3' and 5′-GGATGGAGTACATATTATT-3') versus control siRNA

  • CRISPR/Cas9 knockout: Generate UTP11 knockout cell lines as definitive negative controls

  • Overexpression: Transfect cells with Flag-tagged UTP11 expression constructs and confirm co-localization with the antibody signal

• Peptide competition assays:

  • Pre-incubate antibody with recombinant UTP11 protein or immunogenic peptide

  • Valid antibodies will show reduced or eliminated signal after competition

• Western blot validation:

  • Confirm detection of protein at expected molecular weight (~28 kDa)

  • Verify detection of UTP11 in known high-expressing cancers versus low-expressing normal tissues

• Cross-reactivity assessment:

  • Test antibody against proteins with similar domains (other UTP family members)

  • Confirm signal correlation across multiple detection methods (IF, WB, IP)

For UTP11 specifically, validation should include immunostaining to confirm nucleolar localization, consistent with its function in rRNA processing.

What are the most effective troubleshooting strategies for non-specific binding when using UTP11 HRP-conjugated antibodies?

When encountering non-specific binding with UTP11 HRP-conjugated antibodies:

• Optimize blocking conditions:

  • Use 5% BSA instead of milk for blocking phospho-epitopes

  • Add 0.1-0.5% Tween-20 to reduce hydrophobic interactions

  • Include 10% normal serum from the same species as the sample to block Fc receptors

• Sample preparation refinements:

  • Extend fixation time for better epitope preservation

  • Perform antigen retrieval optimization (citrate buffer pH 6.0 vs. EDTA buffer pH 8.0)

  • Filter lysates to remove aggregates that cause non-specific binding

• Antibody dilution and incubation adjustments:

  • Test higher dilutions (1:1000-1:5000) to reduce non-specific binding

  • Reduce incubation temperature from room temperature to 4°C

  • Add 0.1% non-homologous protein (like fish gelatin) to antibody diluent

• Signal development modifications:

  • Shorten substrate incubation time to minimize background development

  • Use substrate with lower sensitivity for high-abundance targets

  • Include 1-5 mM sodium azide in wash buffers to inhibit endogenous peroxidases

For nucleolar proteins like UTP11, additional high-salt washes (300-500 mM NaCl) can help reduce non-specific nucleic acid-mediated interactions.

What conjugation methods are recommended for preparing custom UTP11 HRP-conjugated antibodies?

Several conjugation methods are available for researchers preparing custom UTP11 HRP-conjugated antibodies:

• Heterobifunctional crosslinker method:

  • Use Sulfo-SMCC to generate maleimide-activated HRP

  • Create sulfhydryl groups on UTP11 antibodies through SATA-mediated thiolation

  • The reaction forms stable thioether bonds between antibody and HRP

  • Advantages: Controlled reaction conditions, minimal antibody aggregation

• Site-directed photocrosslinking:

  • Mix UTP11 antibody with photoactivatable HRP conjugate

  • Illuminate with 365nm light (6-10W) for site-specific conjugation to heavy chains

  • Results in uniform labeling of 1-2 HRP molecules per antibody

  • Advantages: Rapid protocol (under 2 hours), minimal hands-on time (30 seconds)

• Rapid conjugation kits:

  • Commercial kits contain pre-activated HRP and optimization reagents

  • Directional covalent bonding ensures proper orientation

  • Compatible with small quantities of antibody at near-neutral pH

  • Advantages: High conjugation efficiency, 100% antibody recovery

The choice of method depends on laboratory resources, expertise, and the specific application requirements for the UTP11 antibody conjugate.

What buffer conditions maximize stability and performance of UTP11 HRP-conjugated antibodies?

Optimal buffer conditions for UTP11 HRP-conjugated antibodies include:

• Storage buffer composition:

  • 50% Glycerol in PBS (pH 7.4) provides cryoprotection

  • Addition of 0.01M PBS maintains physiological conditions

  • 0.03% Proclin 300 prevents microbial contamination

  • 1-5 mM EDTA chelates metal ions that could degrade the antibody

• Working buffer considerations:

  • For Western blotting: TBS-T (20mM Tris, 150mM NaCl, 0.1% Tween-20, pH 7.6)

  • For ELISA: PBS-T (10mM phosphate, 150mM NaCl, 0.05% Tween-20, pH 7.4)

  • For IHC: TBS with 0.025% Triton X-100

• Protein stabilizers:

  • 1% BSA prevents adsorption to container surfaces

  • 1-5 mM sodium azide inhibits microbial growth (not compatible with HRP activity assays)

  • 0.1-1.0 mM PMSF protects against serine proteases

• Temperature considerations:

  • Store concentrated stock at -20°C

  • Working dilutions stable at 4°C for 1-2 weeks

  • Avoid repeated freeze-thaw cycles (aliquot before freezing)

For UTP11 specifically, antibody performance may be enhanced in buffers containing 1mM DTT to maintain reducing conditions that better expose the epitope.

What controls are essential when performing UTP11 detection experiments with HRP-conjugated antibodies?

A comprehensive control strategy for UTP11 detection should include:

• Antibody controls:

  • Isotype control: IgG from same species as UTP11 antibody, HRP-conjugated

  • Secondary antibody only control (for indirect detection methods)

  • Peptide competition control: UTP11 antibody pre-incubated with immunizing peptide

• Sample controls:

  • Positive control: Cell line with known UTP11 overexpression (e.g., hepatocellular carcinoma)

  • Negative control: UTP11 knockdown samples using validated siRNA (5′-GAAGCTAAGAAAATCGAAA-3')

  • Normal tissue control: Tissues with baseline UTP11 expression

• Technique-specific controls:

  • For Western blotting: GAPDH or β-actin loading control

  • For IHC: Adjacent tissue sections with primary antibody omitted

  • For IP experiments: Non-specific IgG immunoprecipitation

• Biological validation controls:

  • RPL5 and RPL11 co-detection to confirm nucleolar stress pathway activation

  • p53 and p21 detection to verify downstream signaling

  • NRF2 and SLC7A11 measurement to confirm ferroptosis pathway involvement

Given UTP11's role in nucleolar stress, comparing results under conditions that induce nucleolar stress (e.g., low-dose actinomycin D treatment) provides additional validation.

How can I optimize experimental protocols for studying UTP11's role in cancer using HRP-conjugated antibodies?

For comprehensive investigation of UTP11's oncogenic functions:

• Expression analysis in cancer tissues:

  • HRP-IHC protocol:

    • Antigen retrieval: 10mM citrate buffer, pH 6.0, 95°C, 20 min

    • Antibody dilution: 1:200-1:500 in TBS + 1% BSA

    • Development: DAB substrate, monitor for 2-10 minutes

    • Counterstain: Hematoxylin for nuclei visualization

• Nucleolar stress pathway analysis:

  • Co-immunoprecipitation protocol:

    • Lyse cells in buffer containing 20mM Tris-HCl, 150mM NaCl, 1% NP-40, 1mM EDTA, pH 7.4

    • Immunoprecipitate UTP11 with specific antibody

    • Probe for interactions with MPP10, RPL5, and RPL11

    • Detect with appropriate HRP-conjugated antibodies

• p53-dependent growth arrest analysis:

  • Western blot protocol:

    • Harvest cells 48-72h after UTP11 siRNA transfection

    • Separate proteins on 10-12% SDS-PAGE

    • Transfer to PVDF membrane at 100V for 1h

    • Block with 5% BSA in TBS-T

    • Probe with HRP-conjugated antibodies against UTP11, p53, MDM2, and p21

• Ferroptosis pathway analysis:

  • RNA immunoprecipitation protocol:

    • Transfect cells with Flag-UTP11

    • Lyse in RIP buffer (10mM Tris, 150mM NaCl, 1mM Na2EDTA, 0.1% SDS, 1% NP-40)

    • Immunoprecipitate with anti-Flag magnetic beads

    • Extract RNA and perform RT-qPCR for NRF2 and SLC7A11 transcripts

These optimized protocols leverage the sensitivity of HRP-conjugated antibodies while addressing the specific biological questions surrounding UTP11's role in cancer.

What is the recommended protocol for western blot analysis of UTP11 using HRP-conjugated antibodies?

For optimal western blot detection of UTP11:

Sample preparation:

• Lyse cells in RIPA buffer (50mM Tris pH 7.4, 150mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS)

• Include protease inhibitors (PMSF, aprotinin, leupeptin)

• Sonicate briefly to shear DNA and break nucleoli

• Centrifuge at 14,000g for 15 minutes at 4°C to clear lysate

Gel electrophoresis and transfer:

• Load 20-50μg protein per lane on 12% SDS-PAGE

• Include molecular weight markers covering 20-30kDa range

• Transfer to PVDF membrane at 100V for 60 minutes or 30V overnight

Antibody incubation:

• Block membrane with 5% BSA in TBS-T for 1 hour at room temperature

• Incubate with UTP11 HRP-conjugated antibody (1:1000 dilution) overnight at 4°C

• Wash 4 times for 5 minutes each with TBS-T

Detection:

• Apply chemiluminescent substrate and incubate for 1 minute

• Expose to X-ray film or capture using digital imaging system

• Expected band: ~28kDa for native UTP11

Controls to include:

• Positive control: Hepatocellular carcinoma cell lysate

• Negative control: UTP11 knockdown sample

• Loading control: GAPDH or β-actin

For studying UTP11 interactions with MPP10 or ribosomal proteins, co-immunoprecipitation followed by western blotting provides valuable insights into complex formation.

How can I use UTP11 HRP-conjugated antibodies to study the mechanisms of ferroptosis in cancer cells?

To investigate UTP11's role in ferroptosis using HRP-conjugated antibodies:

• Expression correlation analysis:

  • Perform western blots on cancer cell panels to correlate UTP11 expression with:

    • NRF2 protein levels

    • SLC7A11 expression

    • Glutathione (GSH) levels (measured biochemically)

  • Use HRP-conjugated antibodies for UTP11, NRF2, and SLC7A11 detection

• UTP11 knockdown experiments:

  • Transfect cells with UTP11 siRNA (5′-GAAGCTAAGAAAATCGAAA-3')

  • At 48-72h post-transfection, analyze:

    • UTP11 protein levels by western blot

    • NRF2 mRNA stability using actinomycin D chase

    • SLC7A11 protein expression

    • Lipid peroxidation (using BODIPY-C11 staining)

    • Cell death (using annexin V/PI staining)

• RNA immunoprecipitation (RIP) protocol:

  • Transfect cells with Flag-UTP11

  • Lyse in RIP buffer containing RNase inhibitors

  • Immunoprecipitate UTP11-RNA complexes with anti-Flag beads

  • Extract RNA and analyze NRF2 transcripts by RT-qPCR

  • Compare to input and IgG controls

• Rescue experiments:

  • In UTP11-depleted cells, test rescue with:

    • NRF2 overexpression

    • SLC7A11 overexpression

    • GSH supplementation

    • Ferrostatin-1 (ferroptosis inhibitor)

  • Analyze by western blotting with HRP-conjugated antibodies

This approach combines protein detection with functional assays to establish the mechanistic link between UTP11 and ferroptosis in cancer cells.

What protocol modifications are required when using UTP11 HRP-conjugated antibodies for immunohistochemistry?

For effective immunohistochemical detection of UTP11:

Tissue preparation:

• Fix tissues in 10% neutral buffered formalin for 24-48 hours

• Process and embed in paraffin

• Section at 4-5μm thickness

• Mount on positively charged slides

Antigen retrieval optimization:

• Compare heat-induced epitope retrieval methods:

  • Citrate buffer (pH 6.0)

  • EDTA buffer (pH 8.0)

  • Tris-EDTA (pH 9.0)

• Microwave or pressure cooker treatment for 15-20 minutes

Primary antibody protocol:

• Quench endogenous peroxidase with 3% H₂O₂ for 10 minutes

• Block with 5% normal serum in PBS for 1 hour

• Apply UTP11 HRP-conjugated antibody at 1:50-1:200 dilution

• Incubate overnight at 4°C in humidified chamber

• Wash 3×5 minutes with PBS

Signal development:

• Apply DAB substrate and monitor for 2-10 minutes

• Counterstain with hematoxylin for 30-60 seconds

• Dehydrate through graded alcohols and clear in xylene

• Mount with permanent mounting medium

Special considerations for UTP11:

• Include nucleolar markers (nucleolin or fibrillarin) on serial sections

• Use low-dose actinomycin D-treated tissues as positive controls for nucleolar stress

• Consider dual immunofluorescence with p53 to correlate UTP11 levels with p53 activation

These modifications optimize detection of UTP11's nucleolar localization and expression changes in cancer tissues.

How can I design experiments to investigate the relationship between UTP11 and p53 using HRP-conjugated antibodies?

To explore the UTP11-p53 regulatory axis:

• Co-expression analysis in tissue panels:

  • Perform IHC on serial sections of cancer tissues for:

    • UTP11 (HRP-conjugated antibody)

    • p53 (HRP-conjugated antibody)

    • MDM2

    • p21

  • Quantify expression correlation using digital image analysis

• UTP11 knockdown effects on p53 pathway:

  • Transfect cells with UTP11 siRNA

  • Harvest at multiple timepoints (24h, 48h, 72h)

  • Analyze by western blot for:

    • UTP11 depletion

    • p53 accumulation

    • MDM2 expression

    • p21 induction

  • Include both p53+/+ and p53-/- cell lines to distinguish p53-dependent effects

• p53 ubiquitination assay:

  • Transfect p53-/- cells expressing shNC or shUTP11 with:

    • p53 expression plasmid

    • HA-MDM2 plasmid

    • His-Ub plasmid

  • Treat with proteasome inhibitor MG132 (4-6h)

  • Perform Ni-NTA pulldown of His-tagged proteins

  • Analyze ubiquitinated p53 by western blot

• Ribosomal protein-MDM2 interaction:

  • Immunoprecipitate MDM2 from control and UTP11-depleted cells

  • Probe for RPL5 and RPL11 co-precipitation

  • Use specific siRNAs against RPL5 (5′-GGAGGAGAUGUAUAAGAAA-3') and RPL11 (5′-GGAACUUCGCAUCCGCAAA-3') to verify pathway

This experimental design leverages the sensitivity of HRP-conjugated antibodies to detect both high-abundance (MDM2) and low-abundance proteins (ubiquitinated species) within the same pathway.

How should I design xenograft studies to evaluate UTP11 function using HRP-conjugated antibodies for analysis?

For comprehensive xenograft studies of UTP11 function:

Study design:

• Animal model: 4-week-old female BALB/c nude mice

• Cell lines:

  • CAL-51 cells stably expressing shNC or shUTP11 (6×10⁶ cells)

  • HCT116 p53+/+ and HCT116 p53-/- cells with shNC or shUTP11 (5×10⁶ cells)

• Injection: Subcutaneous in right flank, suspended in DMEM with 50% Matrigel

• Measurements: Tumor volume calculation using formula (length × width²) × 0.52

• Duration: Monitor until control tumors reach ~1000mm³

Tissue analysis protocol:

• Harvest and process tumors:

  • Fix one portion in 10% neutral buffered formalin for IHC

  • Snap-freeze another portion for protein/RNA extraction

  • Prepare single-cell suspensions for flow cytometry

• Immunohistochemical analysis with HRP-conjugated antibodies:

  • UTP11 expression (verify knockdown efficiency)

  • p53 accumulation and activation

  • p21 expression (cell cycle arrest marker)

  • Ki-67 (proliferation marker)

  • Cleaved caspase-3 (apoptosis marker)

  • 4-HNE staining (lipid peroxidation/ferroptosis marker)

• Western blot analysis of tumor lysates:

  • UTP11, p53, p21, MDM2 protein levels

  • RPL5 and RPL11 expression

  • NRF2 and SLC7A11 levels

• Correlation analysis:

  • UTP11 expression vs. tumor volume

  • p53 status effect on tumor growth inhibition

  • Markers of ferroptosis vs. UTP11 levels

This design allows evaluation of both p53-dependent and p53-independent mechanisms of UTP11 in tumor growth.

How can I interpret conflicting data when UTP11 HRP-conjugated antibody results differ from other detection methods?

When facing discrepancies between UTP11 HRP-conjugated antibody results and other methods:

• Epitope accessibility analysis:

  • Different antibodies may recognize distinct epitopes

  • The UTP11 epitope sequence (HIIKETKEEVTPEQLKLMRTQDVKYIEMKRVAEAKKIERLKSELHLLDFQGKQQNKHVFFFDTKKEVEQFDVATHLQTAPEL) may be differentially accessible

  • Test multiple antibody clones targeting different regions of UTP11

  • Compare native vs. denatured detection methods

• Post-translational modification interference:

  • Some antibodies may be sensitive to phosphorylation, acetylation, or other modifications

  • Treat samples with phosphatases or deacetylases before analysis

  • Compare results from different cellular compartments (cytoplasmic vs. nuclear fractions)

• Cross-reactivity assessment:

  • Test antibody specificity against recombinant UTP11

  • Perform immunoprecipitation followed by mass spectrometry

  • Compare results in UTP11 knockout/knockdown models

• Technical validation approach:

  • Systematically vary protocol conditions:

    • Fixation methods (paraformaldehyde vs. methanol)

    • Detergent concentrations (0.1-1.0% Triton X-100)

    • Buffer compositions (phosphate vs. Tris-based)

  • Use orthogonal detection methods (fluorescence vs. HRP)

  • Verify mRNA expression by RT-qPCR or in situ hybridization

When properly validated, discrepancies often reveal biologically relevant information about protein conformation, interactions, or modifications in different contexts.

What experimental approaches can distinguish between UTP11's roles in nucleolar stress and ferroptosis pathways?

To dissect UTP11's dual roles in nucleolar stress and ferroptosis:

• Genetic separation-of-function experiments:

  • Generate domain-specific UTP11 mutants:

    • N-terminal mutants that disrupt MPP10 binding

    • C-terminal mutants that affect mRNA binding

  • Rescue UTP11-depleted cells with these mutants

  • Assess nucleolar stress markers and ferroptosis independently

• Pathway-specific inhibition:

  • Block p53 activation using pifithrin-α

  • Inhibit ferroptosis using ferrostatin-1 or liproxstatin-1

  • Supplement glutathione using N-acetylcysteine

  • Analyze growth inhibition and cell death in UTP11-depleted cells under each condition

• Time-course experiments:

  • Monitor temporal sequence after UTP11 depletion:

    • 0-24h: Nucleolar morphology changes and rRNA processing

    • 24-48h: p53 stabilization and p21 induction

    • 48-72h: NRF2 mRNA degradation and SLC7A11 reduction

    • 72-96h: Lipid peroxidation and ferroptotic cell death

• Selective pathway activation:

  • Induce nucleolar stress using low-dose actinomycin D

  • Trigger ferroptosis using erastin or RSL3

  • Compare effects with UTP11 depletion

  • Use HRP-conjugated antibodies to detect pathway-specific markers