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. 2016 Sep 23;7(43):70364–70377. doi: 10.18632/oncotarget.12217

Figure 3. Stabilization of ZEB1 mRNA via its 3′-UTR by Beclin 1 knockdown in FRO cells.

Figure 3

(A) Newly synthesized RNA was labeled and captured using Click-iT Nascent RNA Capture Kit (Life Technology), and nascent ZEB1 mRNA was analyzed using Quantitative PCR. (B) Cells were treated with actinomycin D for the indicated time, ZEB1 mRNA was analyzed using Quantitative PCR. ZEB1 mRNA levels were normalized to GAPDH mRNA and plotted as a percentage of the value at time zero from three independent experiments. (C) Schematic representation of the luciferase reporter vector bearing the ZEB1 3′-UTR. (D) FRO cells expressing scramble or shRNA were co-transfected with the luciferase reporter vector bearing the ZEB1 3′-UTR or a control luciferase reporter vector and a Renilla reporter vector. The reporter activity was assessed at 48 h post-transfection. (E) FRO cells expressing scramble or shRNA were transfected with the luciferase reporter vector bearing the ZEB1 3′-UTR for 48 h, then treated with actinomycin D for the indicated time. Luciferase mRNA was analyzed using Quantitative PCR, normalized expression level was plotted as a percentage of the value at time zero from three independent experiments. Similar data was obtained from three independent cell preparations. N.S., not significant; *P < 0.01.