Extended Data Fig. 1: Skein-like inclusion formation does not rely on the RNA binding capacity of TDP-43, but the RRM1 and low complexity domains are required.

(a) Representative images of cells expressing TDP-43^{∆NTD-Clover} treated with NaAsO₂ for 1 to 4 hours. Cells are stained with G3BP1 to show stress granules from more than three biological replicates. An arrow indicates a cell with TDP-43^{∆NTD-Clover} in both stress granules and skein-like inclusions at 2 hours of arsenite treatment. (b-e) Representative live images of cells expressing TDP-43^{90-414-Clover} from more than three biological replicates (b), TDP-43^{90-414-2KQ-Clover} from more than three biological replicates (c), TDP-43^{102-414-2KQ-Clover} from more than three biological replicates (d), TDP-43^{NLSm-S48D-Clover} from more than three biological replicates (e) before and after 4 hours of 100 µM NaAsO₂ treatment. Black-white inverted fluorescent images are shown. n = 4 biological replicates representing 149, 214, 146, 215 cells (TDP-43^NLSm-Clover) and 37, 55, 58, 37 cells (TDP-43^{NLSm-S48D-Clover}), respectively. Error bar: SD. (f) FRAP analysis of TDP-43 skein-like inclusions induced by MG132 from more than three biological replicates. High magnification images show no fluorescence recovery of photo bleaching points within 1 min. (g) Representative images of MG132-induced skein-like TDP-43 inclusions preserved after mild cell permeabilization. (h-i) Representative live images of cells expressing TDP-43^{174-414-Clover} (h) and TDP-43^{∆NTD-∆320-343-Clover} (i) before and after 4 hours of 100 µM NaAsO₂ treatment from more than three biological replicates. Black-white inverted fluorescent images are shown. N = 4 biological replicates for TDP-43^{NLSm-S48D-Clover}. (j) Representative images of cells expressing TDP-43^∆NTD-V5 under conditions of no stress, NaAsO₂, or MG132 treatment from more than three biological replicates. TDP-43^∆NTD-V5 is indicated by V5 staining.

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