Extended Data Fig. 4: Myo-granules in skeletal muscle contain TDP-43 and are amyloid-like oligomers. | Nature

Extended Data Fig. 4: Myo-granules in skeletal muscle contain TDP-43 and are amyloid-like oligomers.

From: TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle

Extended Data Fig. 4

Related to Fig. 2. a, C2C12 myotubes differentiated for seven days reveal strong A11 immunoreactivity in MHC+ myotubes, but no A11 immunoreactivity in undifferentiated PAX7+ myoblasts. n = 3 independent experiments. Scale bar, 50 μm. b, Muscle stem cells isolated from four-month-old C57/BL6 mice were differentiated in culture for five days and show cytoplasmic and nuclear expression of A11 oligomers. Myotubes express MHC. Scale bar, 50 μm. n = 3 mice. c, Deconvolution microscopy of C2C12 myotubes differentiated for seven days reveal punctate A11 staining in MHC+ myotubes, but no A11 signal was found in undifferentiated myoblasts. Scale bar, 25 μm. n = 3 independent experiments. d, Secondary antibody-only control for A11 staining in tibialis anterior muscle sections at 5 DPI. Nuclei were counterstained with DAPI. Scale bar, 25 μm. n = 4 mice. e, Representative images of A11 and TDP-43 co-localization in tibialis anterior muscle for uninjured muscles, and at 5 DPI and 10 DPI. Scale bars, 25 μm. n = 3 mice. f, Secondary antibody-only control for A11–TDP-43 co-localization in tibialis anterior muscle sections at 5 DPI and 10 DPI shows a lack of signal. Nuclei were counterstained with DAPI. Scale bar, 25 μm. n = 3 mice. g, Quantification of A11 signal intensity in myofibres from e. Unpaired, two-tailed Student’s t-test; comparison between uninjured muscle and 5 DPI, ****P = 4.4 × 10−5; comparison between 5 DPI and 10 DPI, ***P = 4.1 × 10−4; comparison between 10 DPI and uninjured muscle P = 0.024 (P value not shown). n = 3 mice per condition, n = 10 myofibres were averaged per mouse. Data are mean ± s.d. h, Representative deconvolution image of A11 immunoreactivity and eMHC expression in the mouse tibialis anterior myofibres at 5 DPI that were quantified in Fig. 2c. n = 3 mice, each showing similar results. Scale bars, 2 μm and 0.8 μm (inset). i, Proximity ligation assays reveal complexes of TDP-43 and A11 (green) in C2C12 myotubes counterstained with phalloidin (red). A PLA positive control with two antibodies that recognize different epitopes of TDP-43 are positive, whereas complexes are absent if one primary antibody is omitted. n = 3 independent experiments per condition. Ms, mouse; Rb, rabbit.

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