Single- and double-knockdown of WT1 and GATA4 in XX and XY gonads.

<p>(A-D) XX and XY gonads were dissected from murine embryos (12.5 dpc) and incubated for 72 h with <i>Wt1</i> and/ or <i>Gata4 vivo</i>-morpholinos. Transfection with appropriate mismatch <i>vivo</i>-morpholinos served as a negative control. For double-knockdown of WT1 and GATA4, <i>vivo</i>-morpholinos were applied simultaneously. (A) Efficient knockdown of WT1 and/or GATA4 was assessed by immunoblot analysis. (B-D) Relative transcript levels were determined by qRT-PCR and normalized to <i>Sdha</i> transcripts according to the 2^ΔΔCt method [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0176296#pone.0176296.ref034" target="_blank">34</a>]. Results are shown as fold differences between cultures treated with mismatch <i>vs</i>. <i>Wt1-</i> (B) and mismatch <i>vs</i>. <i>Gata4-</i>morpholino (C), respectively. Panel D represents the effect of the double-knockdown of WT1 and GATA4. Error bars represent S.E.M. from independent biological replicates (n ≥ 5). scale = log2. Statistical differences are indicated by asterisks: *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001, ****<i>p</i><0.0005, paired <i>t</i>-test. n.d., not detectable.