Figure 3.

Multivalent GU-oligonucleotides attenuate transcriptional-blockade-induced TDP-43 mislocalization and accelerate nuclear re-import

(A) Schematic of the nuclear retention assay, in which oligonucleotides were first transfected into HeLa cells, incubated for 5 h, and TDP-43 mislocalization induced by NVP2 transcriptional blockade (250 nM for 1 h), prior to fixation and TDP-43 immunostaining.

(B) Representative TDP-43 immunofluorescence images, nuclear retention assay. Scale bar: 20 μm.

(C–F) TDP-43 N/C (% mock-transfected cells, i.e., Lipofectamine only) in (GU)16- (C), Clip34nt- (D), (‘AUG12’)2- (E), or (CA)16 (F)-transfected cells with or without NVP2 treatment. Mean ± SD of ≥7 biological replicates. Note: -NVP2 curves are the same as in Figure 2D. See also Figures S2 and S3.

(G) Schematic of the nuclear re-import assay, in which TDP-43 mislocalization was first induced by NVP2 treatment (250 nM for 1 h), followed by NVP2 washout and oligonucleotide transfection. A subset of cells were fixed immediately after NVP2 treatment and at time points up to 8 h for TDP-43 immunostaining.

(H) Representative TDP-43 immunofluorescence images, nuclear re-import assay. Scale bar: 20 μm.

(I) TDP-43 N/C normalized non-NVP2-treated cells at each time point. Mean ± SD of three biological replicates.

(J) Non-linear regression analysis of time to 80% recovery (minutes); 95% confidence intervals are shown.

In (B, H) the intensity histogram for each image was independently spread between the dimmest and brightest pixels, and a pseudo-color linear LUT covering the full range of the data was applied (see legend).

In (C–F) NS = not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001 by two-way ANOVA with Tukey’s post-hoc test.