Fig. 4.

Reactivation of the Xi-linked Mecp2 gene by small-molecule XCIF inhibitors. (A and B) Two-color RNA FISH monitoring expression of Xist (red) and Mecp2 (green) in differentiated mouse ES cells treated with DMSO (control or –), OSU-03012, or LY294002 (A), and in BMSL2 cells treated with DMSO or GNE-317 (B). Representative images are shown (Upper) using the higher concentrations of the inhibitors, and the results quantified (Lower). The yellow arrowheads indicate colocalizing Xist and Mecp2 signals; the white arrowheads indicate Mecp2 signals not colocalizing with Xist. (C) Two-color RNA FISH monitoring Xist (red) and Mecp2 (green) expression in mouse cortical neurons treated with DMSO (control or –), OSU-03012, BX912, or LY294002. Representative images are shown (Upper), and the results quantified (Lower). The arrowheads indicate Mecp2 signals. (D) Two-color RNA FISH monitoring Xist (red) and Mecp2 (green) expression in BMSL2 cells treated with DMSO (control or –), LY294002, or OSU-03012, and at least 6 d following removal of the inhibitor. Representative images are shown (Upper), and the results quantified (Lower). The arrowheads indicate Mecp2 signals. (E) qRT-PCR monitoring Xi-linked wild-type MECP2 expression in human RTT fibroblasts treated with DMSO (–), 5-azacytidine (5-AZA), BX912, OSU-03012, or VX680. As a control, Xa-linked wild-type MECP2 expression was monitored in another clonal fibroblast cell line derived from the same RTT patient (lane 1). The arrowhead indicates the wild-type MECP2 qRT-PCR product. GAPDH was monitored as a loading control. (Lower) Schematic of the MECP2 wild-type (wt) and mutant (mut) alleles.