Fig. 3: Loss of miR106a-RepA pairing interferes with Xist-Xi association.
A qRT-PCR analysis monitoring Xist levels in H4SV cells expressing miR106sp following treatment with actinomycin D. Gapdh mRNA was used as a normalization control. n = 3. For 0 h, *p = 0.014; 2 h, **p = 0.0013; 4 h, **p = 0.0089. B Schematic showing the in vitro stability assay for P32-labeled RepA in whole-cell lysates expressing NS, miR106sp, or miR106a mimics (Left). Slot blot assay monitoring stability of in vitro synthesized uncapped RepA in a time-dependent manner. In vitro-synthesized Gapdh is used as an endogenous control. The representative images (Middle) and the results quantified from three experiments (Right) are shown. For RepA, 24 h. miR106a mimics vs. NS, ***p = 0.00049; miR106sp vs. NS, ***p = 0.00035; miR106sp vs. miR106a mimics ***p = 9.39 × 10-6. C qRT-PCR analysis of EU-labeled nascent Xist RNA prepared from the cells expressing control or miR106sp. Gapdh mRNA was used as a normalization control. n = 3. For 0 h, **p = 0.0091; 2 h, **p = 0.0061; 4 h, **p = 0.0052. D, E Confocal-airyscan sections of nuclei stained for Xist in control and miR106sp- (D) or LNAs (E) treated H4SV cells. The proportion of Xist-positive cells is quantitated (Right). The representative images are shown, and the results are quantified from three experiments D, E. Data is expressed as mean ± SD (A–C). Unpaired, two-sided t-test with no multiple adjustments (A–C). Scale bars: 2 µm (D, Left) and 5 µm (D, Right).
