Extended Data Fig. 6: G3BP1 assembles into stress granules in aged neurons.

a, Volcano plot of G3BP1 pulldown and mass spectrometry of all detected proteins in unstressed versus stressed iPSC-diff.1 neurons (n = 2 replicates). P values were calculated using a two-tailed Welch’s t test. b, Scatter plot of G3BP1 pulldown fold change plotted with the proteomic fold change for each detected protein (n = 2 replicates). c, Scatter plot of TDP-43 pulldown fold change versus G3BP1 pulldown fold change for all proteins falling within the spliceosome KEGG term (hsa03040). d, Quantification of the number of stress granules per cell in the indicated isogenic cell types (n = 6 replicates). The box plots represent the 25th to 75th percentile, the centerline denotes the median and the error bars denote the range of all data values. Statistics were calculated using a two-tailed Welch’s t test. e, G3BP1 and DAPI staining of various transdifferentiated neuron lines visualized by fluorescence confocal microscopy. The yellow box denotes the inset. White scale bar = 10 μm; yellow scale bar = 2 μm. f, Same as d, but for the additional Tdiff lines from e. Tdiff.1 was replotted from d. Statistics were calculated pairwise with the Tdiff.Y1 line using a two-tailed Welch’s t test. g, Fluorescence confocal images of primary fibroblasts stained for stress granule markers. White scale bar = 10 μm. h, Quantification of the average stress granule area, circularity and roundness in stressed Tdiff.1 (Fig. 2c/ED6I) versus iPSC-diff.1 neurons (Extended Data Fig. 3f) plotted as in ED6D (n > 100 stress granules in N = 5 replicates). Statistics were calculated using a two-tailed Welch’s t test. i, Confocal fluorescence images of unstressed and arsenite-stressed transdifferentiated neurons stained for G3BP1 (green) and poly(A) (magenta) via a dT FISH probe. Yellow boxes denote the inset region. White scale bar = 10 μm; yellow scale bar = 2 μm.