<i>APTR</i> regulates <i>p21</i> epigenetically via the PRC2 complex.

<p>(A) Q-RT-PCR shows induction of <i>p21</i> mRNA normalized to <i>GAPDH</i> after <i>EZH2</i> or <i>SUZ12</i> knockdown on the left. Fold change compared to si<i>GL2</i>-transfected cells. Mean ±s.e.m, n>6, **: P = 0.006. ***: P<0.0001). (B) Endogenous EZH2 and SUZ12 were analyzed by immunoblot after EZH2 or SUZ12 knockdown as a control for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095216#pone-0095216-g005" target="_blank">Figure 5A</a>. (C) Schematic of <i>p21</i> promoter and primer pairs for CLIP PCR (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095216#pone-0095216-g003" target="_blank">Figure 3F</a>). Boxes: binding of indicated proteins/RNA in control cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095216#pone-0095216-g003" target="_blank">Figure 3F</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095216#pone-0095216-g005" target="_blank">5E–F</a>). Grey areas: regions where binding is downregulated by <i>siAPTR#2</i>. (D) qRT-PCR shows <i>APTR</i> knockdown efficiency by <i>siAPTR#2</i> in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095216#pone-0095216-g005" target="_blank">Figure 5E–G</a>. (E–F) ChIP of SUZ12 or H3K27me3 on the <i>p21</i> promoter in 293T cells transfected by either <i>siGL2</i> or <i>siAPTR#2</i>. X-axes: primer-pairs 1–7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095216#pone-0095216-g003" target="_blank">Figure 3F</a>. Y-axes: %Input values were presented. Mean ±s.e.m. n = 6. (G) ChIP of SUZ12 or H3K27me3 on the <i>HOXD11</i> locus in 293T cells transfected by either <i>siGL2</i> or <i>siAPTR#2</i>. X- axis: antibodies used for ChIP. Y-axis: % input of <i>HOXD11</i> locus in precipitates. (n.s.: not significant, Mean ±s.e.m. n = 6) (H) CLIP of MS2-<i>APTR</i> or <i>MS2</i> RNA alone in the <i>p21</i> promoter (primer-pair 2 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095216#pone-0095216-g003" target="_blank">Figure 3F</a>) and the <i>HOXD11</i> locus. Mean ±s.e.m. n = 6. Y-axis shows amount of specific DNA in the precipitate normalized to that in the CLIP of <i>MS2</i> alone. (I) CLIP of <i>MS2</i>-<i>APTR</i> or <i>MS2</i> RNA alone on the <i>p21</i> promoter in 293T cells transfected by si<i>GL2</i> or si<i>EZH2</i>. X-axes: primer-pairs 1-7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095216#pone-0095216-g003" target="_blank">Figure 3F</a>. Y-axis: amount of specific DNA in the precipitate normalized to that in CLIP of <i>MS2</i> alone. Mean ±s.e.m. n = 3.