Extended Data Fig. 10: Expression of MCP–CIZ1 or MCP–GFP–MCP does not rescue phenotypes due to loss of the E-repeat.

a, RNA FISH images of Tsix transcripts for detection of two X chromosomes. Two ΔE MCP–CIZ1 ES cell clones (9 and 10) are shown. b, Immunoblot result for undifferentiated ΔE ES cell clones expressing MCP–CIZ1. c, Representative epifluorescence images of day 7 differentiated MCP–CIZ1-expressing ΔE clones, probed for Xist and MS2. d, Proportion of Xist clouds also displaying a co-localizing MS2 signal at differentiation day 7. The results for both CIZ1 rescue clones from one experiment were merged and the error bars represent s.e.m (n = 120), P: not significant, two-tailed Student’s t-test. e, Quantification of nascent Gpc4 or Atrx expression patterns in wild-type, ΔE, or ΔE cells expressing MCP–CIZ1 (clone 9) displaying Xist^MS2 expression, at differentiation day 7 (n = 50, from one experiment). See k for legend. f, Representative epifluorescence images of in wild-type, ΔE or indicated ΔE rescue cell lines at differentiation day 7 immunostained for CIZ1 and probed for MS2. Arrowheads indicate rescued cloud from the ΔE Xist^MS2 allele. Fraction of MS2+Xist clouds showing CIZ1 enrichment is given. g, RNA FISH images of Tsix transcripts in ΔE MCP–GFP–MCP ES cells to demonstrate the presence of two X chromosomes. h, Representative epifluorescence images of day 7 differentiated MCP–GFP–MCP-expressing ΔE ES cells probed for Xist and MS2, and illustration of Flag-tagged MCP–GFP–MCP fusion protein (see Fig. 3b for key). i, Immunoblot against the Flag-tag and GAPDH using lysates from undifferentiated MCP–GFP–MCP ΔE ES cells. j, Histogram showing the proportion of nuclei with Xist FISH signal that also displayed a co-localizing MS2 signal at differentiation day 7 for indicated cell lines (n = 100); P: not significant, two-tailed Student’s t-test, using 2 independent MCP–GFP–MCP expressing clones from one experiment. k, Quantification of nascent Gpc4 or Atrx expression patterns in cells displaying Xist^MS2 expression at differentiation day 7 (n = 50, from one experiment). For images in b and i, see Supplementary Fig. 1 for source data.