Research Paper Volume 4, Issue 10 pp 695—708

Figure 5. Effect of NF90 on the expression of reporter constructs bearing SASP 3'UTRs. (A) Schematic of reporters used. Reporter constructs were derived from the psiCHECK2 parent plasmid by insertion of 3'UTRs from CCL2, CCL16, TNFRSF11B, CSF2, CXCL1, IL8, and IL6 mRNAs (gray) in the 3' end of the renilla luciferase (RL) coding region (dark blue); the internal transfection control firefly luciferase (FL, light blue) was expressed from the same plasmid backbone. (B) Biotin pulldown analysis of the interaction of NF90 with the biotinylated 3'UTRs shown in panel A (gray) was carried out as explained in the Methods section. Negative control RNAs spanned the 5'UTR and CR of the nucleolin (NCL) mRNA. (C)Left, WI-38 cells (pdl 30) were transfected with Ctrl or NF90 siRNAs; 24 h later, cells were further transfected with each of the plasmids shown [WI-38 cells (800 ng/ml plasmid) and IDH4 cells (8 ng/ml plasmid)] and 16 h after that, the levels of Renilla luciferase were calculated and normalized to the levels of Firefly luciferase in the same transfection group. Right, the “translation indeces” were calculated for the reporter constructs. Relative differences in RL/FL were compared with differences in RL mRNA/FL mRNA, in order to calculate how much of the changes in reporter activity were due to changes in transcript levels (relative to changes in protein levels). (D) IDH4 cells were transfected and investigated using the same strategies as described in panel C. In (C,D) data are the means +SD from three independent experiments.