Fig. 2.

Reactivation of the Xi induced by shRNAs. (A) Firefly luciferase activity induced by shRNAs in Xi-8 cells normalized to shControl. For each graph in A–D a control is plotted followed by an average of three measurements for each shRNA in ascending order, with SD indicated by error bars. P < 0.05 for all hairpins; individual measurements for each shRNA are provided in Dataset S2. Rank order of shRNAs is provided on the x axis to facilitate identification of individual hairpins from Dataset S2 in the graphs. (B) Firefly luciferase activity induced by shRNAs in Xi-8 cells in combination with 0.2 µM 5-AZA normalized to the control shRNA in combination with 0.2 µM 5-AZA. At this concentration, the treatment of 5-AZA did not induce a measurable increase in luciferase activity in shControls. (C) Reactivation of the native MeCP2 gene on the inactive X in Xa-3 cells by shRNAs in combination with 0.2 µM 5-AZA. RT-qPCR of MeCP2 mRNA in Xa-3 cells containing different shRNAs in combination with 0.2 µM 5-AZA normalized to shControls in combination with 0.2 µM 5-AZA, measured using the primers indicated by red lines. (D) Reactivation of the CMV-luciferase reporter at the Hprt locus on the inactive X by shRNAs in combination with 0.2 µM 5-AZA. Firefly luciferase activity induced by shRNAs in CMV-FL cells in combination with 0.2 µM 5-AZA normalized to control shRNA in combination with 0.2 µM 5-AZA. (E) Allele-specific RT-PCR for the PGK1 gene on the Xi in M. spretus/M. musculus hybrid Patski cell line demonstrates reactivation using a combination of shRNA and 0.2 µM 5-AZA. The knockout efficiency expressed as the fraction remaining was as follows: Bmpr2, 0.33; XIST, 0.24; Clspn, 0.26; Sgk2, 0.19; Prkd3, 0.28; Erb4, 0.32; Plk2, 0.23; Parp6, 0.19; Arid4b, 0.50; Dtx3l, 0.37; Msl2l1, 0.25; Smrcd2, 0.24; Ttn, 0.42; and Acss1, 0.27. Expression levels in shRNA-containing cells treated with 0.2 µM 5-AZA are compared with shControl cells treated with 0.2 µM 5-AZA (P < 0.05 for all hairpins), error bars indicate SD.