Extended Data Fig. 11: CELF1 enhances droplet formation of PTBP1 with the E-repeat in vitro and mutations in MATR3 and TDP-43 that abrogate their self-association do not rescue ΔE phenotypes.

a, Images showing lack of droplets with 60 μM rPTBP1, 3.2 μM E-repeat or control RNA at 40 min. b, Droplets formed from 60 μM rPTBP1 and 0.5 μM E-repeat RNA over time. c, Same as a except with 0.5 μM control RNA and different concentrations of rPTBP1 (40 min). d, Same as a except with 60 μM rPTBP1 and 20 μM rCELF1, or 38 μΜ rCELF1 with 0.5 μM E-Repeat RNA. Arrowheads indicate solution boundary with sample on left. e, Bright-field images showing aggregate-like formations of 20 μM rCELF1, 0.5 μM E-repeat RNA with varied concentrations of rPTBP1. f, RNA FISH images of Tsix transcripts in indicated ES cell lines to show presence of both X chromosomes. g (i), MATR3 immunoblot on extracts from ΔE ES cells expressing MCP–MATR3(S85C). (ii), TDP-43 immunoblot on ΔE ES cells expressing MCP–TDP-43(EGGG). h, Epifluorescence images of day 7 differentiated ΔE cells expressing MCP–MATR3(S85C) (i) or MCP–TDP-43(EGGG) (ii) probed for Xist and MS2. i, MEFs (Xist^2lox/2lox, R26^{M2rtTA/tetO-Cre}) probed for Xist, before or after dox treatment (96 h). Percentage of cells with displayed Xist pattern is given (n = 50, two biological replicates). j, Histogram showing percentage of MEFs with H3K27me3 or CELF1 Xi-enrichment under conditions described in i. Error bars represent s.e.m, (n = 50, from two biological replicates). k, Histogram showing relative Xist abundance over time of dox treatment for cells in i (see Fig. 4g.). l, Experimental schematic for knockdown experiment in m–q. m, Immunoblot showing knockdown of indicated factors in the experiment described in l. n, Percentage of MEFs (no dox) with an Xist cloud for indicated knockdowns (n = 50, from one experiment). o, Percentage of MEFs (no dox) with an Xi-enrichment of H3K27me3 that show a co-localizing accumulation of CELF1 (n = 50, from one experiment). p, Same as n except with dox treatment. q, Percentage of MEFs with CELF1 enrichment (n = 50, from one experiment). For images in g and m, see Supplementary Fig. 1 for source data.