Extended Data Fig. 3: Myo-granules isolated from cells and mice contain TDP-43 and are amyloid-like oligomers.
From: TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle
Related to Fig. 2. a, b, X-ray diffraction of immunoprecipitated myo-granules (right half of a, b) compared to the diffraction of mock IgG immunoprecipitation (left half a) and to the diffraction of super oxide dismutase 1 (SOD1) amyloid oligomers (left half of b). For all diffraction patterns, two rings at approximately 4.8 Å and approximately 10 Å are drawn on the bottom half to highlight the absence of an approximately 4.8 Å reflection in the mock immunoprecipitation and a similar approximately 4.8 Å reflection with the absence of an approximately 10 Å reflection in the SOD1 diffraction. One sample per condition was used. Two diffraction images at different rotations were taken per sample and each image gave similar results. c, d, Complexes that were immunopurified using TDP-43 (c) or A11 (d) were isolated from C2C12 myotubes. Complexes express A11 (c) and TDP-43 (d), whereas immunopurified TDP-43 or A11 myo-granules that were immunostained with secondary antibodies only lack signal. Red, TDP-43 or A11 immunoreactivity. n = 3 independent experiments. Scale bars, 1 μm. e, f, Complexes that were immunopurified using TDP-43 (e) or A11 (f) were isolated from tibialis anterior muscle at 5 DPI. Complexes express A11 (e) and TDP-43 (f), whereas immunopurified TDP-43 or A11 myo-granules immunostained with secondary antibodies only lack signal. Red, TDP-43 or A11 immunoreactivity. n = 3 mice. Scale bars, 0.05 μm. g, TDP-43 immunopurified complexes isolated from an uninjured tibialis anterior muscle (contralateral to the 5 DPI muscle) reveal no complexes with an A11 oligomeric confirmation. n = 3 mice. Scale bars, 0.05 μm. h, A11 immunopurified complexes from an uninjured tibialis anterior muscle (contralateral to the 5 DPI muscle) reveal no complexes containing TDP-43. n = 3 mice. Scale bars, 0.05 μm. i, Dot blot of A11 immunoreactivity in C2C12 cells differentiated into myotubes compared to myoblasts. Quantification reflects fold change in dot blot signal from myoblast to myotube. Data are mean ± s.d., n = 3 independent experiments. j, k, Quantification of the dot blot signal for A11 conformation complexes (j) and TDP-43 conformation complexes (k) during skeletal muscle regeneration at 5 DPI and 10 DPI compared to contralateral uninjured tibialis anterior muscle and normalized to the HRP-only signal. Quantification reflects fold change in dot blot signal. Data are mean ± s.d., n = 3 mice, P values were obtained using unpaired, two-tailed Student’s t-tests.
