Exploratory experiment designed to discover new patterns targeting SQSTM1, CALCOCO2 in knockout cell lines. Primary outcome: stress granule accumulation upon receptor knockout
Functional validation of autophagy receptors SQSTM1/p62 and CALCOCO2/NDP52 in stress granule elimination through single knockout experiments. The study demonstrated that knockout of either receptor individually causes accumulation of both physiological and pathological stress granules, establishing their essential roles in stress granule clearance. This provides evidence that autophagy-mediated elimination is a critical mechanism for maintaining stress granule homeostasis.
Cell lines: Generate SQSTM1-/- and CALCOCO2-/- knockouts in U2OS cells via CRISPR-Cas9. Use dual gRNA strategy (2 gRNAs per gene) to delete coding exons. Clone and validate single-cell clones by: (1) Western blot (no protein detectable). (2) Sanger sequencing (frameshift or deletion). (3) Off-target analysis (sequence top 5 predicted off-targets). Maintain WT parental line as control. Stress granule assays: (1) Physiological SG: 0.5 mM sodium arsenite (1h), or 200 μM puromycin (1h, translation inhibitor). (2) Pathological SG: Transfect with ALS-linked mutants (TDP-43 M337V, FUS R521C) or OPMD mutant (PABPN1 A17). (3) Recovery: Induce SG with arsenite, wash out, follow for 0-8h. Quantification: Immunostain for G3BP1 (SG marker), count SG per cell (n=100 cells per genotype per condition).
...Quantitative predictions: (1) WT cells: Arsenite induces 15-20 SG per cell, which clear by t1/2=3-4h after washout. By 8h, <10% of cells retain SG. (2) SQSTM1-/-: SG count 20-25 per cell (slightly more form), t1/2=6-7h (60% slower clearance). At 8h, 40-50% cells still have SG. (3) CALCOCO2-/-: Similar to SQSTM1-/-, t1/2=5-6h, 30-40% cells with SG at 8h. (4) Pathological SG (TDP-43 M337V): WT cells clear ~50% of SG by 24h. KO lines clear <20% (severe accumulation). (5) Re-expression of cognate receptor: Rescues clearance to near-WT levels (t1/2 within 1h of WT, p>0.1).
...Primary: SQSTM1-/- or CALCOCO2-/- increases SG persistence >1.5-fold vs. WT (t1/2, log-rank test p<0.01). At 8h post-washout, >30% cells retain SG in KO vs. <10% in WT (p<0.01, chi-square). Secondary: (1) Pathological SG (disease mutants) accumulate more severely in KO lines (>2-fold retention vs. WT, p<0.01). (2) Receptor re-expression rescues phenotype (clearance kinetics not significantly different from WT, p>0.05). (3) Bafilomycin phenocopies KO (p>0.1 for WT+bafilomycin vs. KO). (4) LC3-II accumulation on SG fractions reduced >40% in KO (p<0.01).
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