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 experiment tests the hypothesis that G3BP proteins contribute to TDP-43 pathological aggregation through mechanisms independent of canonical stress granule formation. While G3BP1/2 are well-established stress granule nucleators, recent evidence suggests they may also facilitate protein aggregation through direct protein-protein interactions or alt

METHODOLOGY NOTES

**Phase 1: Cell Culture Preparation (Days 1-3)** • Maintain HEK293T, SH-SY5Y, and primary cortical neurons in appropriate media • Transfect cells with TDP-43 wildtype, A315T, and M337V constructs using Lipofectamine 3000 • Generate stable G3BP1/G3BP2 knockout cell lines using CRISPR-Cas9 (n=3 clones per line) • Validate knockout efficiency by Western blot and qRT-PCR **Phase 2: G3BP Inhibition and TDP-43 Expression (Days 4-7)** • Treat cells with G3BP inhibitors: C108 (10-50 μM), ISRIB (200 nM), or siRNA knockdown • Express TDP-43 constructs for 24-48 hours to induce aggregation • Avoid stress conditions (no arsenite, heat shock, or oxidative stress) • Monitor cell viability using MTT assay and trypan blue exclusion **Phase 3: TDP-43 Aggregation Analysis (Days 8-10)** • Fix cells at 24, 48, and 72 hours post-transfection • Perform immunofluorescence using anti-TDP-43 (1:500) and anti-G3BP1 (1:200) antibodies • Quantify cytoplasmic TDP-43 aggregates >1 μm diameter per cell (n≥200 cell