Atf7ip is required for Xi maintenance and acts synergistically with DNA methylation. (A) Representative immunostaining images of MEFs treated with control knockdown (in this case targeting a sequence within GFP) or Atf7ip knockdown for 72 h and stained for ATF7IP (green). Nuclei were detected with DAPI stain. (B) Western blot of cells treated as in (A) with antibody against ATF7IP (band size approximately 220 kDa), with alpha-tubulin loading control. (C) Quantification of band intensity of the western blot shown in (B), normalized to alpha-tubulin loading control. (D) (Left) Schematic of the X chromosomes in female reporter MEFs carrying (i) the Xi-linked luciferase (XiCAG-LuciferaseXaΔXist MEFs), (ii) H2B-Citrine (XiCAG-H2B-CtirineXaΔXist MEFs), or (iii) GFP (XiCAG-GFPXaΔXist MEFs). (Right) Graphs summarizing the reporter assays for respective MEFs treated with control siRNA treatment (siScramble), siRNAs targeting Atf7ip and Dnmt1, and/or 5-aza-2’-dC (0.2 uM) (also referred to as low concentration 5-aza-2’-dC) as indicated, for 72 h. Error bars indicate standard deviation of raw ALU values (for luciferase) or fluorescent cell count (for GFP and H2B-citrine) from three individual wells with the same treatment from one representative experiment. * = P <0.01 by Student’s T-test. (E) RNA FISH for Xist and the X-linked gene Atrx in MEFs 72 h after addition of 0.2 um (low) 5-aza-2’-dC and transfection of siRNAs targeting Atf7ip. Atrx pinpoints of transcript signal mark the site of nascent transcription. Representative image for a cell displaying expression of Atrx from the Xi, with two pinpoints for Atrx, one associated with the Xist RNA cloud. (F) Summary of counts of nuclei analyzed by RNA FISH for Atrx and Xist RNA, with the indicated FISH patterns.