(A) FISH analysis of p21 in a HNSCC TMA. Green indicates p21 and red denotes the control loci (scale bar 5μm). (B) Relative p21 expression data obtained from cancer genome browser (N-43, T-521) (S4 Table). (C) Relative mRNA quantity of p21 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 FXR1 and p21 levels estimated by qRT-PCR in UMSCC74B cells after treatment with FXR1 shRNA. Cells were collected at indicated time points. FXR1 and p21 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 p21 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 GAPDH 3′UTR plasmid or different segments of P21 3′UTR, the lysates were analyzed for luciferase activity using luminometer. The empty 3’UTR luciferase plasmid and luciferase-fused GAPDH 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 p21seg1 and p21seg2RNAs at the G4 region. RNP IP was performed 48 h post-transfection of UMSCC74B cells with seg1 and seg2 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. (*p<0.05, **p<0.005, ***p<0.0005).

https://doi.org/10.1371/journal.pgen.1006411.g001