<i>APTR</i> suppresses <i>p21</i> transcription.

<p>(A) Q-RT-PCR shows induction of <i>p21</i> mRNA (normalized to <i>GAPDH</i>) after <i>siAPTR</i>. Fold change compared to <i>siGL2</i>-transfected 293T cells (mean ±s.e.m., n>6, ***: P<0.0005). (B) The induction of p21 protein in the si<i>APTR</i>#2 transfected 293T cells is prevented by overexpression of sense but not antisense <i>APTR</i>. Fold change of p21 normalized to ACTIN, compared to si<i>GL2</i>-transfected cells (mean ±s.e.m., n>3, **: P<0.005). (C) Growth suppression after <i>siAPTR</i> is alleviated in <i>p21</i>^−/− HCT116 cells. Mean ±s.e.m. n = 9. Right: <i>APTR</i> expression levels (Normalized to <i>GAPDH</i>) measured by Q-RT-PCR in <i>p21</i>^+/+ or <i>p21</i>^−/− HCT116 cells at the indicated days after transfection of siRNAs (n.s.: not significant, ****: P<0.0001). (D) Q-RT-PCR shows fold change of <i>APTR</i> (normalized to GAPDH), compared to the si<i>GL2</i>-transfected cells in the two cell lines in C (mean ±.e.m., n = 3). Note that cells were transfected on Day 1, so Day 2 is 1 day after transfection and Day 6 is 5 days after transfection. Thus si-<i>APTR</i> does not decrease <i>APTR</i> on day 1 after transfection, but the <i>APTR</i> RNA remains low up to day 5. (E) Schematic of MS2-CLIP. The dark line is <i>APTR</i> RNA fused to MS2 binding sequences. (F) <i>APTR</i> associates with the <i>p21</i> promoter. <b>Top</b>: The % of input DNA present in the MS2BP-YFP CLIP is shown in cells expressing <i>MS2</i> alone or <i>MS2</i>-<i>APTR</i> (mean ±s.e.m, n>6). 1–7 refer to the primer pairs in the schematic. <b>Bottom</b>: locations of <i>p21</i> promoter fragments amplified by primer pairs 1–7 in the CLIP assay. Grey bar: area where <i>APTR</i> binds.