🧫
DDR factor depletion and focus formation analysis
active
experiment
Created: 2026-04-06T12:30:33
By: etl-v1-backfill
Quality:
50%
✓ SciDEX
ID: exp-f2ee282d-3319-4ba2-a2e0-ab792e0bec32
🧫 Experiment Protocol
ExploratoryMED1, CDK9cultured cells and in vitro nucleosome systemproposed
This experiment examined the role of transcriptional machinery components in DNA damage response (DDR) focus formation through loss-of-function approaches. The researchers used absence or inactivation of key factors (likely RNA polymerase II, MED1, CDK9) and measured the resulting effects on DDR foci formation. The study employed both in vivo cellular systems and an in vitro reconstitution system using nucleosomes to demonstrate that these transcriptional factors are necessary for proper DDR focus assembly. This approach allowed direct testing of whether transcriptional machinery is required for the spatial organization of DNA repair factors into discrete foci.
PRIMARY OUTCOME
Reduction in DDR foci formation
EXPECTED OUTCOMES
## Primary Outcomes
**Focus Formation Impairment**: MED1 knockdown reduces IR-induced γH2AX focus formation by ≥40% (p < 0.001) and delays 53BP1 focus resolution (50% resolution time: 6h vs. 2h in controls). CDK9 knockdown produces similar but milder effects (-25% γH2AX focus number).
**Promoter Occupancy**: MED1 and CDK9 occupy DDR gene promoters (p21, PUMA, GADD45) at baseline and increase 3-5-fold upon DNA damage. Knockdown reduces promoter occupancy by ≥50%, confirming direct transcriptional regulation.
## Secondary Outcomes
**Rescue Specificity**: Wild-type MED1 rescue restores γH2AX focus formation to ≥80% of control levels, confirming on-target specificity. Catalytically inactive MED1 mutant fails to rescue, indicating CDK9 kinase activity requirement.
**Temporal Dynamics**: MED1/CDK9 knockdown specifically impairs late-stage DDR (6-24h timepoints) while early DSB detection (30 min) remains intact, suggesting role in repair factor recruitment rather than initial recognition.
SUCCESS CRITERIA
## Primary Success Criteria
**Knockdown Efficiency**: Both siRNAs per target must achieve ≥65% protein reduction vs. non-targeting siRNA control (western blot densitometry), confirmed in ≥3 independent experiments.
**Focus Phenotype**: DDR factor knockdown must produce ≥25% reduction in IR-induced γH2AX or 53BP1 focus count at ≥1 timepoint (6h or 24h post-IR) with statistical significance (p < 0.05, n≥3 independent experiments).
## Secondary Success Criteria
**ChIP Confirmation**: MED1/CDK9 occupancy at DDR gene promoters must show ≥2-fold enrichment over IgG control at baseline, increasing to ≥4-fold upon DNA damage (validates direct recruitment to damage-responsive genes).
**Rescue Requirement**: Rescue cDNA must restore focus formation to ≥70% of control levels, confirming phenotype specificity.
PROTOCOL
# DDR Factor Depletion and Focus Formation Analysis Protocol
## Phase 1: siRNA Knockdown of DDR Factors in Cell Culture (Days 1-21)
**Cell Line Selection**: Use U2OS cells (osteosarcoma,具有良好的 DNA 损伤修复能力) for initial experiments. Maintain in DMEM + 10% FBS at 37°C, 5% CO₂. Authenticate via STR profiling, test for mycoplasma.
**siRNA Transfection**: Design 3 independent siRNAs per target (MED1/CD247, CDK9, and additional DDR factors: ATM, ATR, BRCA1, RNF8). Transfect U2OS at 50% confluence using RNAiMAX (Life Technologies, 30 nM siRNA, 1:1 ratio). Include non-targeting siRNA pool and untreated controls. Validate knockdown via qRT-PCR at 48h and 72h.
**Immunoblot Validation**: Collect protein at 72h post-transfection. Probe with anti-MED1 (1:500, Abcam #ab36702), anti-CDK9 (1:500, Abcam #ab79416), and loading control anti-β-actin (1:5000). Quantify via Li-COR Odyssey CLx (IR fluorescence). Target ≥70% knockdown at protein level.
## Phase 2: DNA Damage Induction and Focus Formation Assay (Days 22-42)
**DNA Damage Induction**: 24h after transfection, induce DNA damage via: (a) ionizing radiation (IR, 2 Gy, X-ray, 5 min), (b) UV-C (20 J/m²), or (c) bleomycin (5 μg/mL, 1 hour). Collect cells at 0.5h, 2h, 6h, 24h post-damage for focus analysis.
**Immunofluorescence Focus Staining**: Pre-extract cells (0.5% Triton X-100 in PBS, 5 min, 4°C) to remove soluble proteins. Fix in 4% PFA (15 min, RT). Block with 5% BSA. Stain primary antibodies: anti-γH2AX (1:500, Millipore #05-636), anti-53BP1 (1:300, Novus Biologicals #NB100-304), anti-MED1 (1:200), anti-CDK9 (1:200). Add Alexa Fluor 488/594 secondary antibodies. Mount with DAPI.
**Image Acquisition and Analysis**: Acquire on Zeiss LSM 880 confocal (63× oil, NA 1.4). For each condition, acquire 15 z-stacks (0.5 μm steps). Count foci per nucleus using ImageJ (particle analysis, threshold 3× background). Score ≥100 cells per condition per experiment.
## Phase 3: ChIP-qPCR and Functional Rescue (Days 43-63)
**Chromatin Immunoprecipitation**: Perform ChIP for MED1 and CDK9 at DDR gene promoters (p21/CDKN1A, BBC3/PUMA, GADD45). Cross-link cells (1% formaldehyde, 10 min), sonicate (Bioruptor, 30 sec on/off, 10 cycles). Immunoprecipitate with anti-MED1 or IgG control. Purify DNA, analyze via qPCR (primers flanking p53 response elements).
**Rescue Experiment**: Clone wild-type MED1 and CDK9 cDNA into pCMV-3xFLAG vector (silent mutations to resist siRNA). Co-transfect with siRNA to test rescue of focus formation phenotype. Assess by immunofluorescence as above.
**Statistical Analysis**: Compare focus counts between knockdown and control via Student's t-test or Mann-Whitney U (for non-normal distributions). Report as mean ± SEM.
Source: PMID 31570834 ↗
🧫 Experiment Extras
PATHWAY
DNA damage response pathway
MARKET PRICE
$0.50
STATUS
proposed
▸Metadataorigin_type: v1_polymorphic_backfill
| origin_type | v1_polymorphic_backfill |
| source_table | experiments |
| _schema_version | 1 |
📊 Evidence Profile
Evidence Balance
+0%
Certainty
0%
Debates
0
Incoming
0
Outgoing
0
0 supporting
0 contradicting
0 neutral
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