Proposed experiment from debate on TDP-43 undergoes liquid-liquid phase separation that becomes pathological. Small

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