Table 1. Estimates for the amount of excess nuclear TDP-43 that is required to induce exon skipping across different experimental conditions.

Our results suggest that an increase in TDP-43 that is greater than 1.1 to 1.5 fold over normal protein levels may lead to exon skipping toxicity. Models for TDP-43 overexpression should avoid increasing nuclear TDP-43 and aim to strictly limit excess TDP-43 to only the cytoplasm.

Excess TDP-43 required to induce exon skipping
Fold change increase nuclear TDP-43 over baseline endogenous	Exon skipping
1.1–1.5x	Miminum exon skipping
1.5–2x	50–100% exon skipping
>2x	Maximal exon skipping
Estimated diffusion of TDP-43NLSm (△NLS TDP-43) into the nucleus
Experimental condition	Percentage of total TDP-43NLSm
Immunoblot of FACS isolated nuclei from transfected cells	10–45%
Immunoblot of inducible iGFP-WT and iGFP-NLSm cells	45%
Fold change of GFP fluorescence intensity by FACS	2–5%*

^*GFP is indirect measurement and may not accurately estimate the amount of nuclear △NLS-TDP-43