Extended Data Fig. 1: Depletion of PTBP1, MATR3, CELF1 and TDP-43 does not strongly affect gene silencing during the Xist-dependent stage of XCI initiation.

a, Experimental schematic. b (i), Immunoblot confirming the siRNA-mediated knockdown of PTBP1, MATR3, and CELF1, normalized to GAPDH. Asterisks indicate non-specific bands. (ii), As in (i), except for TDP-43. Error represents the s.e.m. from three independent experiments. For source data see Supplementary Fig. 1. c (i), Graph showing nascent transcription patterns of the X-linked gene Gpc4 after 3 days of differentiation and knockdown of the indicated factor (spot refers to nascent transcription event on one chromosome) (n = 50 from 1 experiment). (ii), Same as (i) but for Chic1. (iii), Same as (i) but for Atrx. d, Representative images of siRNA-treated differentiating cells immunostained for indicated proteins (red), probed for Xist (green) and DAPI stained (blue). e (i), Schematic for aggregation score calculation. (ii), Box plots showing Xist aggregation scores upon depletion of indicated proteins. Independent siGFP controls were used for CELF1 and TDP-43 experiments. (iii), Box plots showing the Xist mask values used to calculate the aggregation scores in (ii). (iv), Box plots showing the bounding circle area values encompassing the Xist mask used to calculate the Xist aggregation scores in (ii). For box plots in (ii)–(iv): (n = 25): *P < 0.05, ***P < 0.0005, ****P < 0.00005; two-tailed Kolmogorov–Smirnov test from one replicate in b. Horizontal lines denote the median, whiskers indicate 1.5× the interquartile range, dots represent outliers.