Figure S5.

Three RNA features define the condensation-binding relationships, related to Figure 5

A. Normalized gene expression for the genes with regulated PAS (see Figure 6A) in control and after WT or TDP-43 variant induction.

B. The features of each binding region class, which contribute to their classification. The 36 classes of binding regions are ordered as in Figure 5, and the following features are shown: the number of regions in each class, the average cDNA count per replicate in the ‘HD’ experiment (Table S1), average length of regions in each class, the average density of all evaluated motifs, and the average coverage of YG- or AA-containing [UG]n motifs.

C. A separate experiment was analyzed as explained in Figure 5A of TDP-43 mutant lines at a lower RNaseI concentration (0.2 units per 1 mL of lysate) resulting in longer RNA fragments.

D. A separate experiment was analyzed as explained in Figure 5A, containing WT TDP-43 and 316del346 triplicate samples pre-treated either with 1,6- or 2,5-HD. C and D are linked to Table S3 containing quantification of cDNA counts from CLIP samples overlapping with the binding regions, together with their genomic coordinates, region, gene id and gene names and derived classifications in groups by length, density and base content.

E. Maximum z-projection of confocal z stacks of dox-induced GFP-TDP-43 WT in HEK Flp-In cells after incubation with 8% 2,5-HD or 8% 1,6-HD for 5 minutes.

F. Quantification of foci counts in each segmented nucleus from confocal z stacks. n (counted cells): 2,5-HD = 7, 1,6-HD = 5; segmented from n (fields of view): 2,5-HD = 2, 1,6-HD = 2. Significance was tested with a Welch Two Sample t test (^∗p = 0.017).

G. Turbidity measurements on pre-formed TDP-43-MBP condensates show a reduction in turbidity after addition of 1,6-HD compared to addition of buffer (n = 3, two-tailed t test, p < 0.0001).

H. Alexa488-labeled TDP-43-MBP (1:200 labeled:unlabeled) was used to image TDP-43-MBP condensates after addition of buffer or 1,6-HD. Scale bar, 10 μm.

I. Decrease in area fraction of TDP-43-MBP condensates upon addition of 1,6-HD compared to addition of buffer (n= 15 images, two-tailed t test, p < 0.0001).

J. Decrease in TDP-43-MBP condensate size upon addition of 1,6-HD compared to addition of buffer (n = 15 images, two-tailed t test, p < 0.0001).