Extended Data Fig. 8: Characterization of roX2 in hybrid and tethered MSL2-expressing mES cells.

a, Schematic representation of the experimental setup showing the RNA levels after transcription inhibition with flavopiridol at the indicated time points in cell lines D11 (MSL2^WT) and G2 (MSL2^dCXC-CTD hybrid) expressing roX2 (random, single-copy integration). The RNA levels were determined by RT–qPCR, normalized to a spike that was added before RNA isolation and expressed relative to the 0 min control time point in the D11 (MSL2^WT) cell line. The line plot connects the mean at each time point ± s.e.m. of n = 7 (MSL2^WT) or n = 5 (MSL2^dCXC-CTD hybrid) biological replicates. Nascent RNA levels decrease at timeframes correlating with the length of the analysed gene (for example, Pgk1: 16.6 kb, intron levels drop by 50% after around 15 min). mRNAs (for example, Rplp0, Pgk1), rRNA (RNA Polymerase I) or U6 snRNA (RNA Polymerase III) remain rather unaffected within the timeframe of 15 min. Total roX2 levels (0.5 kb) decrease by 50% within 15 min of flavopiridol inhibition, pointing towards a rapid turnover (also see Fig. 3h). b, Cropped immunoblots comparing the Flag-tagged MSL2^WT (D11) with the MSL2^dCXC-∆CTD-3xFlag-His-λN cell line (clone 513-A6, abbreviated as MSL2∆^λN). The coomassie-stained gel, DHX9 and OCT3/4 serve as loading controls. For source data, see Supplementary Fig. 1. c, Representative RNA FISH pictures of roX2 RNA (red) and DAPI (light blue) in the MSL2^dCXC-CTD hybrid mES cells (clone G2) with stably integrated roX2 (top) or the MSL2^dCXC-∆CTD-3xFlag-His-λN mES cells (clone 513-A6, abbreviated as MSL2∆^λN) with stably integrated 2×boxB-roX2 (bottom). The pictures are single z-planes with scale bars = 10 μm. d, Quantification of RNA FISH in c obtained from 3 independent hybridizations in 2 stable clones. Induction of the roX2 foci in mouse ESCs by either the drosophilized hybrid (MSL2^dCXC-CTD) or by ectopic tethering of the mouse MSL2 to roX RNA (MSL2∆^λN) is compared. Left, dot plots with centre line represent the mean ± s.e.m. foci frequency, where each overlaid data point represents the quantification result of a single tiled image. Each image was quantified as the number of cells that display a confined signal in the nucleus as % of all nuclei identified based on DAPI staining (% of nuclei with foci/total nuclei). The total number of quantified nuclei examined over all experiments was n = 447 or n = 284 cells and is displayed on top of the dot plot. Statistical significance was evaluated by a two-sided Wilcoxon rank-sum test. Right, boxplots representing the area of the roX2 foci obtained from orthogonal projection images. Overlaid data points represent individual foci (n = 140 and n = 85). The P-values were obtained with a two-sided Wilcoxon rank-sum test.