Figure 5.

Cytoplasmic CIRP mutants fail to regulate reporter translation via p27 5′UTR. (A) Microscopy analysis of HEK293T cells overexpressing EYFP-tagged wt and NES-mutants of CIRP. The NES was inserted either at the C-terminus (CIRP-NES) or at the N-terminus (NES-CIRP) of CIRP. Cells were fixed and CIRP localization was analyzed by fluorescence microscopy. Nuclei were stained with Hoechst (blue). Scale bar: 20 μm. (B) HEK293 cells were transfected with pGL3c or pGL5′UTR, a control plasmid encoding EYFP or wt CIRP or CIRP-NES mutant, and Renilla luciferase for normalization. Relative luciferase activity (RLU) was calculated as ratio of firefly and Renilla luciferase activities; pGL3c/control activity was adjusted to 1. Data are shown as mean ± SD, n = 3 independent experiments. Unpaired two-tailed t-test was used to compare the average RLU of pGL5′UTR in the absence or presence of overexpressed CIRP-NES mutant or wt CIRP: *P < 0.05, **P < 0.01.