Figure 2. Xist upregulation from the Xic-TetO remains unaffected upon relocalization to the nuclear lamina.

A) RNA FISH for Xist (red) and Tsix (white) nascent transcripts as well as for processed, accumulating Xist RNA (green) in control and bound PGK12.1 XX_TetO TetR-EGFP-LaminB1 cells on day 4 of differentiation by LIF withdrawal. Depicted are representative cells for each condition. B) Quantification of Xist expression by presence of nascent Xist RNA as focal signal as depicted in A) in control and bound PGK12.1 XX_TetO TetR-EGFP-LaminB1 cells in ESCs and throughout differentiation by LIF withdrawal (n = number of cells). C) Quantification of Xist RNA accumulation by presence of Xist RNA clouds/domains as depicted in A) in control and bound PGK12.1 XX_TetO TetR-EGFP-LaminB1 cells in ESCs and throughout differentiation by LIF withdrawal. In ESCs no Xist RNA domains could be detected and only the presence of small clusters of Xist RNA was scored (n = number of cells). D) Simultaneous RNA/DNA FISH for Xist RNA (red) and Xpr (X pairing region, BAC 5 (474E04), white) and the TetO array locus (green) performed on control PGK12.1 XX_TetO TetR-EGFP-LaminB1 cells on day 4 of differentiation by LIF withdrawal. Xist RNA is stable during denaturation and can be detected after performing DNA FISH. Depicted is a representative example in which Xist RNA became up-regulated from the wildtype Xic and started coating the wildtype X chromosome. E) Quantification of the distribution of up-regulation of Xist from the Xic-TetO or the wildtype Xic in control and bound PGK12.1 XX_TetO TetR-EGFP-LaminB1 cells on day 4 of differentiation by LIF withdrawal based on simultaneous RNA/DNA FISH as depicted in D) (n = total number of cells; mean results of three experiments; error bars indicate standard deviation; for B,C: one representative experiment is depicted in the quantification).