Extended Data Fig. 4: CELF1 and PTBP1 localize within the Xist-coated territory.

a, Experimental schematic. b, Left, confocal-Airyscan sections of wild-type ES cells at differentiation day 3 and 7, immunostained for CELF1 and H3K27me3. Inset, enlargement of the Xi territory. Right, CELF1 staining in greyscale. c, Histogram showing the proportion of H3K27me3-marked Xi’s with a co-localizing CELF1 enrichment. Error bars indicate s.e.m. (n = 50 from 3 coverslips across 2 independent differentiations); *P < 0.05, two-tailed Student’s t-test. d (i), Intensity values for CELF1 fluorescence were recorded across a 2 μm line over the Xi (identified on the basis of the H3K27me3 Xi-staining) or within the nucleoplasm of the same nucleus in z-stack projections. (ii), Box plot showing the distribution of the ratio between the top 10% CELF1 Xi intensity values compared to the top 10% intensity values from the nucleoplasm (n = 12, from one experiment); *P < 0.05, two-sample Kolmogorov–Smirnov test. e (i), Left, As in b, but showing PTBP1 immunostaining at differentiation day 3. Right, PTBP1 staining in greyscale. (ii), As in (i), except at differentiation day 7. Note that these images highlight a mesh-like PTBP1 concentration within the Xi observed in a small fraction of cells, distinct from that observed in the nucleoplasm of these cells or from the pattern within the Xi at day 3. f (i), As in e (i), but showing MATR3 immunostaining and Xi-zoom ins. (ii), As in e (ii), except showing MATR3 immunostaining. g, As in f, except for TDP-43. h, As in d (ii), except showing data for PTBP1, MATR3 and TDP-43, (n = 5, from one experiment). Red dots, data points for the top 10% Xi/Nucleoplasmic intensity values from 5 cells. For box plots in d (ii) and h, horizontal lines denote the median, whiskers indicate 1.5× the interquartile range, dots represent outliers.