Table 1.

Falsifiable predictions of the lysosomal-stress cargo-fate hypothesis in TNBC.

Prediction	Testable design	Expected interpretation and falsifier
RIPK1 effects extend beyond the Manzamine A model.	Compare pharmacological and genetic RIPK1 perturbation across mechanistically distinct lysosomal stressors and molecularly distinct TNBC models; where feasible, rescue with wild-type or kinase-inactive RIPK1.	Reproducible effects across stressors and models support broader involvement. Restriction to Manzamine A would define a drug- and context-specific role.
A transcriptional mechanism predicts RIPK1-dependent changes in RASAL2 expression.	Use serial RASAL2 mRNA and protein measurements, followed by promoter, chromatin or transcriptional-reporter assays.	An mRNA change preceding protein and phenotype supports transcriptional regulation. Protein-state changes without transcript change argue against it.
A post-translational mechanism predicts changes in RASAL2 protein state without corresponding transcript changes.	Measure RASAL2 phosphorylation, localization, stability and binding partners under stress and RIPK1 perturbation.	A reproducible protein-state change with stable mRNA supports post-translational regulation. No such change weakens this mechanism.
RIPK1 and RASAL2 converge functionally.	Apply single and combined perturbations and compare proximal lysosomal or autophagic readouts with downstream MVB and sEV readouts.	Non-additive effects support a shared limiting process or interaction; additive effects support parallel contributions to a common output; no interaction with distinct proximal effects supports independent arms.
Direct RASAL2-Rab27a coupling, if present, should be demonstrable independently of intact-cell co-immunoprecipitation.	Use cellular co-IP and Rab27a-GTP results as starting evidence (31). Propose purified-protein or domain-mapping binding assays and a direct Rab27a GTP-hydrolysis assay.	Purified-component binding supports direct interaction; accelerated GTP hydrolysis supports direct GAP activity. Negative results favor indirect regulation.
Lysosomal and autophagic changes precede sEV-associated release.	Sample early lysosomal pH, protease activity, membrane integrity and RIPK1-associated changes; intermediate LC3 flux, p62 turnover and autophagosome-lysosome or -MVB relationships; and later Rab27a activity, MVB docking and sEV-associated release.	A pre-death lysosomal/autophagic sequence preceding sEV increase supports cargo-fate switching. Particle increase only after marked injury argues against an active switch.
Regulated EV release is separable from death-associated particles.	At each time point assess Annexin V/PI, LDH, caspase-3/7 or PARP and, where relevant, phosphorylated MLKL (40). Normalize particle output to viable cell number and conditioning time (29).	Particle increase coupled to membrane loss, apoptosis, necrosis or necroptosis supports passive or injury-related release rather than regulated cargo-fate switching.

EV, extracellular vesicle; sEV, small extracellular vesicle; TNBC, triple-negative breast cancer.