FIG 1.

NT2/D1 and HEK293T cells provide the contexts for initiation and maintenance phases of XCI. (A) Semiquantitative RT-PCR for mature and premature XIST using cDNA prepared from HEK293T (female), NT2/D1 (male), and DLD1 (male) cells. 18S rRNA and β-actin serve as controls. (B) qRT-PCR depicting a decrease in the levels of OCT4 and NANOG and increase in PAX6 levels upon RA-mediated differentiation of NT2/D1 cells for 6 days. The x axis represents the differentiation time point, and the y axis represents the fold change normalized to 18S rRNA. Each point on the graph represents values from 5 independent experiments, and error bars represent ±standard errors of the mean (SEM). (C) Immunoblotting showing a decrease in OCT4, SOX2, and NANOG levels upon RA-mediated differentiation of NT2/D1 cells for 6 days. γ-Tubulin serves as an equal loading control. (D) qRT-PCR depicting an increase in the levels of XIST upon RA-mediated differentiation of NT2/D1 cells for 6 days. The x axis represents the differentiation time point, and the y axis represents the fold change normalized to 18S rRNA. Each point on the graph represents values from 3 independent experiments, and the error bars represent ±SEM. (E) X chromosome paint for metaphase spread and interphase nuclei from undifferentiated (0-day) and 5-day RA-treated NT2/D1 cells. RNA FISH for mature XIST for undifferentiated (0-day) and differentiating (5-day, RA) NT2/D1 cells. Arrowheads indicate the FISH signal. (F) Quantification for the RNA FISH signals in 0-day and 5-day RA-treated NT2/D1 cells. n = 3; 200 nuclei were counted for each replicate, and statistical significance was ascertained by Student’s t test.