Fig. 6.

TOP2A increase in G₂/M is influenced by lower miR-548c-3p and higher HuR levels. (A) Fluorescence-activated cell sorter (FACS) analysis of HeLa cells that were synchronized by double thymidine block and released for the times indicated (percentage of cells in each phase is shown). (B to D) Cells treated as described for panel A were used for Western blot analysis to assess the levels of TOP2A, HuR, and control β-actin (B) and for RT-qPCR analysis to quantify the levels of TOP2A mRNA (C) and miR-548c-3p (D). (E) FACS analysis of HeLa cells that were left untreated (asynchronous, Asyn.) or were treated with nocodazole (100 ng/ml, 16 h); the percentage of cells in each cell cycle phase is shown. (F) Lysates were prepared from cells treated as described for panel E, and the levels of TOP2A, HuR, and control β-actin were assessed by Western blot analysis and quantified by densitometry. (G) Total RNA was prepared from cells treated as described for panel E, and the levels of TOP2A mRNA and normalization control GAPDH mRNA were measured by RT-qPCR analysis. (H) The levels of miR-548c-3p were quantified by RT-qPCR (normalized to U6 RNA) in cells that were treated with nocodazole for 16 h (G₂/M group) or released from arrest and tested 8 h (G₁) and 12 h (S) later. (I) HeLa cells transfected with the plasmids shown in Fig. 2C were treated with nocodazole (100 ng/ml), and lysates were prepared 16 h later. The levels of EGFP were assessed and plotted as described for Fig. 2D. (J) Cells were left untreated (Asyn.) or were treated with nocodazole (100 ng/ml); 16 h later, lysates were prepared and the association of TOP2A mRNA with HuR and Ago was measured by RNP IP analysis. (K) Cells were transfected with Ctrl siRNA, HuR siRNA, or miR-548c-3p; 48 h later, cells were either treated with nocodazole (100 ng/ml, 16 h) or left untreated, and TOP2A levels were assessed by Western blot analysis. For panels B to D, n = 2; for panel I, n = 3. *, P < 0.05; **, P < 0.01.