Relationship between Atf7ip and Xist. (A) (i) Representative FISH image for Xist RNA (red) in female DAPI-stained MEFs after 72 h of treatment with 5-aza-2’-dC (0.2 uM) and siRNA-mediated Atf7ip knockdown or GFP (control) knockdown. (ii) As in (i), but displaying an immunostaining image for H3K27me3 under the same conditions. (B) (i) The graph depicts the percentage of DAPI-stained nuclei (n = 200 per sample) with Xi-accumulation of Xist RNA for the conditions shown in (Ai) and additional control treatments. (ii) As in (Bi), but for the Xi-accumulation of H3K27me3. (C) Diagram of the experimental approach to test the effect of combined Xist and Atf7ip depletion on Xi reactivation. Primary female MEFs with an Xi bearing a conditional loxP-flanked allele of Xist (2lox Xist) and a CAG-driven H2B-Citrine transgene in the Hprt locus, and an Xa without a functional Xist gene were obtained by crossing mice with the respective alleles. Cells were treated with either Adenovirus(Ad)-Cre or Ad-empty (empty adenoviral vector) for 24 h, then subjected to Atf7ip or control knockdowns and 5-aza-2’-dC treatments at various concentrations for an additional 72 h, and analyzed by flow cytometry. (D) Representative images of Xist RNA FISH in Ad-Null and Ad-Cre treated MEFs at the time of flow cytometry. The percentage of cells with Xi-like Xist RNA accumulations is given (200 cells counted). (E) Graph representing the percentage of H2B-citrine positive cells after 72 h of the indicated chemical and knockdown treatments. Grey bars indicate the results for Ad-empty treated (Xist-postive) cells and black bars indicate those for Ad-Cre treated (Xist-negative) cells. As siControl we used siRNAs with a scrambled sequence in this experiment.