Figure 6.

AUF1 binding to p21 mRNA is attenuated by circPCNX, but not by mutant circPCNX unable to bind AUF1. (A) Twenty-four hours after transfecting HeLa cells with the plasmids shown, the association of p21 mRNA (or ACTB mRNA, in control reactions) with AUF1 was measured by RIP followed by RT-qPCR analysis and represented as ‘fold enrichment’. (B, C) In HeLa cells transfected with the plasmids indicated, the levels of p21 mRNA (B) were assessed 3 days after transfection by RT-qPCR analysis, and the levels of p21 protein (C) were assessed one and three days after transfection by western blot analysis, including GAPDH as loading control. (D) At the times shown following transfection of HeLa cells with the plasmids indicated, cell proliferation in each transfection group was assessed by using BrdU incorporation analysis. Data in (A, B, D) represent the mean values ± SD from four biological replicates. Significance was established using Student's t-test. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001.