FXR1 destabilizes <i>p21</i> mRNA.

<p>(A) FISH analysis of p21 in a HNSCC TMA. Green indicates p21 and red denotes the control loci (scale bar 5μm). (B) Relative <i>p21</i> expression data obtained from cancer genome browser (N-43, T-521) (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006306#pgen.1006306.s007" target="_blank">S4 Table</a>). (C) Relative mRNA quantity of <i>p21</i> in eight matched HNSCC tumor compared to normal adjacent tissue samples estimated using qRT-PCR. GAPDH serves as a control. (D) Immunoblot analysis of p21 protein from eight representative matched HNSCC tumor and normal adjacent samples. GAPDH is used as a loading control. (E) Relative quantity of <i>FXR1</i> and <i>p21</i> levels estimated by qRT-PCR in UMSCC74B cells after treatment with FXR1 shRNA. Cells were collected at indicated time points. <i>FXR1</i> and <i>p21</i> levels in shControl treated UMSCC74B cells were taken as 1 for each time points. (F) Immunoblot analysis of p21 protein at different time points, as, mentioned in Fig 4E. (G) The mRNA decay rate of <i>p21</i> as indicated in UMSCC74B cells by qRT-PCR after silencing FXR1 followed by transcription inhibition with actinomycin-D for mentioned time points in the graph. Actin serves as a control. Data here represents the mean of n = 3 experiments. (H) Forty-eight hours after transfection of UM74B FXR1 KD and control cells with empty 3’UTR luciferase plasmid, luciferase-fused <i>GAPDH</i> 3′UTR plasmid or different segments of <i>P21</i> 3′UTR, the lysates were analyzed for luciferase activity using luminometer. The empty 3’UTR luciferase plasmid and luciferase-fused <i>GAPDH</i> 3′UTR served as a transfection and loading control. Values are the means ± SD from three independent experiments by using unpaired two sample t-test. (I) Binding of FXR1 with the 3′UTR of <i>p21seg1</i> and <i>p21seg2</i> RNAs at the G4 region. RNP IP was performed 48 h post-transfection of UMSCC74B cells with <i>seg1</i> and <i>seg2</i> 3′UTR fused to a luciferase reporter construct. Luciferase mRNA was detected using qRT-PCR. The luciferase gene in the empty-3′UTR was used as a transfection and qRT-PCR control. (*<i>p<</i>0.05, **<i>p<</i>0.005, ***<i>p<</i>0.0005).