FIGURE 4:

Hsp27 regulates bim-3′UTR sequence during oxidative stress. (A) Quantification of bim mRNA levels bound to the ribosomal protein RPL10a present at the polysome. CGNs were treated with H₂O₂ (37.5 μM). Hsp27 overexpression reduced bim mRNA levels coimmunoprecipitated with RPL10a when compared with controls (*p < 0.05; n = 3). (B) CGNs were cotransfected with a luciferase reporter plasmid containing the bim-3′UTR (divided into two sequences, named 1 and 2) and either pNEO-Hsp27 or a control construct. Bim-3′UTR (sequence 1) neurons showed a significant increase in the luciferase activity after H₂O₂ (37.5 μM) addition (*p < 0.05; n = 3). Coexpression of pNEO-Hsp27 prevented this effect, significantly reducing the luciferase activity (*p < 0.05; n = 3). (C) Bim-3′UTR (sequence 2) neurons did not show any modification of the luciferase activity induced by H₂O₂ treatment or pNEO-Hsp27 coexpression. (D) CGNs were coelectroporated with the luciferase reporter plasmid containing the bim-3′UTR (sequence 1) and either Hsp25 siRNA or control siRNA. Hsp25 siRNA neurons displayed significantly higher luciferase activity than control siRNA neurons (*p < 0.05; n = 3). (E) Quantification of bim mRNA levels bound to the endogenous Hsp25 protein and to the wild-type form of the human Hsp27 (pNEO-Hsp27 construct) overexpressed in CGNs. H₂O₂ (37.5 μM) treatment significantly increased bim mRNA levels coimmuno­precipitated with the Hsp25/Hsp27 compared to control neurons (*p < 0.05; n = 4). The coimmunoprecipitated bim mRNA levels were significantly increased in H₂O₂-treated cells when Hsp27 was overexpressed (*p < 0.05; n = 4). As positive and negative controls for the immuno­precipitation, a specific antibody for Argonaute-2 protein and an unspecific serum were used, respectively. Argonaute-2 levels were detected by Western blot. bim mRNA levels were not detected in the negative control.