Table 4.

Functional assays measuring different import pathways. Overview of the different assays used to measure the effects on nucleocytoplasmic transport. n.a.: not applicable.

Assay	Models	Observations	Suggested Mechanism	Refs
Imp-β1/α1
NLS-NES-GFP	Drosophila salivary glands expressing (G4C2)30 repeats	Reduced N/C ratio	RanGap sequestration in nuclear RNA foci	[107]
	Mutant C9orf72 iPSC-derived neurons	Reduced import based on FRAP
GFP-NLS-NES	U2OS treated with PR20 peptides	Reduced import over time	Pores are blocked by poly-PR peptides	[45]
BSA-NLS	Digitonin-treated HeLa cells exposed to PR20 peptides	Reduced import
RFP-NLSTDP43	HeLa cells transfected with GA149 expressing plasmid	Increased cytoplasmic levels	Cytoplasmic poly-GA aggregates	[100]
	HeLa cells transfected with GR149 expressing plasmid	No consistent differences observed	n.a.
	HeLa cells transfected with PR175 expressing plasmid	No differences observed	n.a.
NES-tdTomato-NLS	Primary mouse cortical neurons expressing TDP-43 CTF/mTDP-43	Reduced N/C ratio	TDP-43 aggregates sequester NC-transport proteins	[90]
	Fibroblasts of mutant C9orf72, TDP-43 and sALS patients
	HEK293T cells transfected with PR50/GR50 expressing plasmids	Mislocalization of reporter	Stress granules sequester NC-transport proteins	[107]
	ALS-FUS human spinal neurons and isogenic controls	Decreased nuclear import	Increased interaction of mutant FUS with Nup62	[99]
Artificial importin- β cargo based on FRET	Permeabilized HeLa cells incubated with PR20 and GR20 peptides	Decreased nuclear import	Poly-PR and poly-GR bind and disrupt cargo loading of importin-β	[47]
Fluorescent dextrans	Permeabilized HeLa cells incubated with PR20 and GR20 peptides	Increased passive transport
Hormone-induced import assay: GCR2-GFP2-TDP43 or GCR2-GFP2-(MBP)-cNLS	HeLa cells incubated with TMR-GR25 peptides	Reduced import	Reduced solubility of importin-α/β via poly-GR binding	[46]
	HeLa cells incubated with TMR-PR25 peptides	No difference observed	n.a.
NLSSV40-mNeonGreen2x-NESpki	HeLa cells incubated with GR20 or PR20 +/− leptomycin B (LMB)	No differences observed	n.a.	[108]
	HeLa cells transduced with GR100 or PR100 +/− LMB	No differences observed	n.a.
	SH-SY5Y cells transduced with GR100 or PR100 +/− LMB	No differences observed	n.a.
	iPSC-derived MNs transduced with GR100 or PR100 +/− LMB	No differences observed	n.a.
NLSc-myc-GFP2x-NESikb2	HeLa cells transduced with GR100 or PR100	No differences observed	n.a.
TPNO
RFP-NLSpY(hnRNPA1)	HeLa cells transfected with GA149, GR149 or PR175 expressing plasmids	No differences observed	n.a.	[100]
YFP-M9-CFP	Digitonin treated HeLa cells incubated with PR20 and GR20 peptides	Decreased nuclear import	Poly-PR and poly-GR bind and disrupt cargo loading of TPNO1	[47]
NESpki-mNeonGreen2x-NLSFUS	HeLa cells incubated with GR20 or PR20	No differences observed	n.a.	[108]