SUMMARY
# Proposed experiment from debate on TDP-43 undergoes liquid-liquid phase separation that becomes pathological. Small
## Background and Rationale
This falsification study critically examines the selectivity of peptide mimetics designed to disrupt pathological TDP-43 liquid-liquid phase separation while preserving physiological TDP-43 functions. TDP-43 proteinopathies are characterized by aberrant phase separation leading to cytoplasmic aggregation, but physiological TDP-43 also undergoes reversi
METHODOLOGY NOTES
**Phase 1: Cell Culture Preparation (Days 1-3)**
• Culture HEK293T and SH-SY5Y neuroblastoma cell lines in DMEM with 10% FBS
• Transfect cells with plasmids encoding wild-type TDP-43, pathological mutants (A315T, M337V), and fluorescently-tagged constructs
• Prepare control cells with empty vector and mock transfection
• Achieve 70-80% confluency in 96-well plates (n=8 wells per condition)
**Phase 2: Peptide Mimetic Characterization (Days 4-6)**
• Synthesize peptide mimetics targeting TDP-43 low-complexity domain interactions
• Prepare serial dilutions from 0.1 μM to 100 μM in serum-free medium
• Perform dose-response curves using alamarBlue viability assay at 24h, 48h, 72h timepoints
• Calculate IC50 values and establish non-toxic concentration ranges
**Phase 3: Phase Separation Selectivity Testing (Days 7-10)**
• Induce physiological TDP-43 condensates using 5% PEG-8000 treatment
• Induce pathological aggregates via oxidative stress (200 μM H2O2) or heat shock (42°C, 2h)
• Apply pe