FIG. 5.

HSP27 increases nuclear NF-κB content, DNA binding, and transcriptional activity in stressed cells. (A) Control-transfected (white bars) and HSP27-transfected (black bars) cells either were left untreated or were treated for 4 h (U937 cells) or 24 h (MEF cells) with etoposide (VP16, 100 μM), ΤNF-α (20 ng/ml), or IL-1β (1 ng/ml). The level of nuclear NF-κB expression was determined by flow cytometry (MFI, mean fluorescence index). (B) Nuclear extracts from control-transfected and HSP27-transfected U937 cells either were left untreated or were treated for 4 h with etoposide (VP16, 100 μM) or ΤNF-α (20 ng/ml) and then were subjected to EMSAs with an NF-κB probe. As a control for binding specificity, the extracts were preincubated with a 50-fold molar excess of unlabeled oligonucleotide (Unl.). Supershift analysis was performed by preincubation of the extracts with an anti-p50 or an anti-p65 polyclonal antibody before EMSAs. (C) Control-transfected (white bars) and HSP27-overexpressing (black bars) cells were transiently transfected with an NF-κB luciferase reporter plasmid and then either were left untreated or were treated for 4 h (U937 cells) or 10 h (MEF cells) with 100 μM VP16 or 20 ng of ΤNF-α/ml. Cotransfection of a thymidine kinase-Renilla luciferase plasmid was used to normalize for transfection efficiency. AU, arbitrary units. Results are the means and standard deviations for three independent experiments.