Extended Data Fig. 2: During muscle formation TDP-43 adopts a higher-order state distinct from stress granules.

Related to Fig. 1. a, Secondary antibody-only control for TDP-43 staining of tibialis anterior muscle sections at 5 DPI. Scale bar, 25 μm. n = 5 mice per condition, representative images are shown, all experiments showed similar results. Nuclei were counterstained with DAPI. b, Representative images of TDP-43 and eMHC immunostaining in tibialis anterior muscle sections at 30 DPI; nuclei were counterstained with DAPI. n = 4 mice. Scale bar, 50 μm. c, RIPA–urea assay reveals the presence of an urea-insoluble TDP-43 fraction isolated from C2C12 myotubes that were differentiated for seven days, but not in C2C12 myoblasts. n = 3 independent experiments, each showing similar results, unpaired, two-tailed Student’s t-test, P = 0.0008. GAPDH remains RIPA-soluble in both myoblasts and myotubes. n = 3 independent experiments, each showing similar results, unpaired, two-tailed Student’s t-test, P = 0.7443. d, Higher molecular weight SDS-resistant TDP-43 assemblies were present in differentiating C2C12 myotubes. Protein assemblies resolved by SDD–AGE. n = 3 independent experiments. Pub1Q/N-GFP from yeast forms SDS-resistant assemblies that have a higher molecular weight than TDP-43 assemblies. e, Schematic of the isolation of myo-granules that contain TDP-43 that are formed during skeletal muscle formation. f, Immunoprecipitation (IP) of TDP-43 on Dynabeads (DB) reveals that oligomers isolated from C2C12 myotubes are absent from myoblasts as observed by TEM. n = 3 independent experiments. g, Stress-granule formation in multinucleated myotubes derived from C2C12 cells. Immunofluorescence using antibodies against stress-granule proteins, G3BP1 and PABP1, after NaAsO₂ treatment or control conditions for 60 min. n = 3 independent experiments, each showing similar results. Zoom, boxed area shown at higher magnification. Scale bars, 5 μm and 20 μm (insets).

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