Related to Fig. 1. a, Nuclear localization of TDP-43 immunofluorescence in C2C12 myoblasts and both nuclear and cytoplasmic localization in C2C12 myotubes differentiated for seven days (n = 3 independent experiment). Myosin heavy chain (MHC) identifies differentiated cells. Scale bars, 25 μm. b, Subcellular fractionation reveals increased cytosolic TDP-43 in differentiating myotubes. Cytosolic (Cyto) myoblasts, 5.0 ± 2.1%; cytosolic myotubes,19.7 ± 3.1%; n = 3 biologically independent experiments that showed similar results, unpaired, two-tailed Student’s t-test, P = 2.0 × 10−3. c, Time course of TDP-43 expression during skeletal-muscle differentiation. n = 3 independent experiments with similar results. Myogenin (MyoG) (magenta) and MHC (green) identify differentiated cells. Nuclei were counterstained with DAPI. Scale bars, 25 μm. d, Top, TDP-43 expression in primary myotubes derived from muscle stem cells that were differentiated in culture for four days. n = 3 independent experiments with similar results. Bottom, images for a secondary-antibody only control. Scale bars, 25 μm. e, Deconvolution microscopy of TDP-43 expression in C2C12 myotubes differentiated for five days. Scale bar, 5 μm. n = 3 independent experiments with similar results. f, CRISPR–Cas9-mediated genomic integration of tetracycline-inducible HaloTag–TDP-43 into the Rosa26 safe-harbour locus in C2C12 myoblasts. A, B and C represent approximate location of primers used in g. g, PCR analyses of gDNA from C2C12 myoblasts for the presence of the HaloTag–TDP-43 construct (top) and integration of the construct into the Rosa26 locus (bottom) using the primers shown in f. n = 3 independent experiments with similar results. Red arrowheads point to the expected PCR product for integration of HaloTag–TDP-43 into Rosa26. Subsequent live-imaging experiments were performed using clones 1 and 4. Non-specific bands are indicated by an asterisk. h, Detection of fluorescently labelled HaloTag–TDP-43 in C2C12 myoblasts following induction resolved on SDS–PAGE. Janelia Fluor 646 (JF646). n = 3 independent experiments with similar results. i, Detection of both HaloTag–TDP-43 and endogenous TDP-43 in selected C2C12 cell clones. n = 3 independent experiments with similar results. j, k, Representative images of individual HaloTag–TDP-43 molecules in a myoblast (j) and a multinucleated myotube (k). Top, start of acquisition (frame 1). Nuclei (Nuc) and cytosolic borders are demarcated by white dotted lines. n = 3 independent experiments with similar results. Bottom, dynamic mapping of single TDP-43 molecule tracks using a multiple target tracing MATLAB script46. Vibrant violet was used to detect myonuclei. Scale bars, 5 μm.