Fig. 7.

Functional analyses of the Zip5 3′-UTR in transfected XEN cells. XEN cells were cultured in ZnA or ZnD media for 30–48 h and then transfected with the luciferase Zip5 3′-UTR reporter constructs. The UTR structure is diagramed above each panel (see Fig. 6 for sequence details). Some ZnD transfected cells had 40 μM zinc added during the last 6–8 h of culture (ZnR). All firefly luciferase values were normalized to co-transfected renilla luciferase values. Each bar represents the mean of 3–4 replicas ± SD. An asterisk indicates a P value <0.05. All sample means were compared to the respective wild-type ZnA mean for the given experiment. a The wild-type (WT) and seed site (SS) mutant UTR vectors were also co-transfected with either an antagomiR vector expressing GFP and siRNA targeting LacZ (αLacZ) or antagomiR vectors expressing GFP and siRNAs targeting miR-193a and miR-328 (α193/328), as described in detail in the “Materials and methods” section. b Concatamerized wild-type Zip5 3′-UTR (WT 3×) and concatamerized Zip5 3′-UTR with scrambled seed sites (SS 3×) were transfected with a GFP expression vector as carrier. c The full length wild-type Zip5 3′-UTR (WT) or the Zip5 3′-UTR with deletion of the stem-loop (SL) were transfected with a GFP expression vector as carrier. Two independent experiments are shown. d The full length wild-type Zip5 3′-UTR (WT) or the Zip5 3′-UTR with both scrambled seed sites and deletion of the putative stem-loop (DM) were transfected with a GFP expression vector as carrier