Figure 5

The ETYA-induced increase in MKP-1 expression involves a HuR-mediated increase in MKP-1 mRNA stability resulting from HuR cytoplasmic translocation. (A) Astrocytes were treated with Act D for the indicated periods in the presence of ETYA, after which MKP-1 mRNA levels were determined by conventional RT-PCR (left, upper panel) and qRT-PCR (right panel). The bar graph represents the intensities of MKP-1 bands normalized against those of GAPDH (left, bottom panel). All data are presented as means ± SDs of three independent experiments (*p < 0.05). (B and C) Astrocytes were transfected with a HuR-specific or nonsilencing control siRNA. Forty-eight h after transfection, cells were stimulated with IFN-γ in the absence or presence of ETYA. MKP-1 mRNA and protein levels were determined by RT-PCR and Western blot analysis (B), and MKP-1 mRNA stability was determined by qRT-PCR (C). The data are presented as means ± SDs of three independent experiments (*p < 0.05 versus IFN-γ + ETYA + con siRNA group). (D) Astrocytes were stimulated with IFN-γ in the presence or absence of ETYA, and nuclear extracts (NE) and cytosolic extracts (CE) were prepared for Western blotting. (E) Confocal microscopic images of astrocytes immunostained with antibodies against HuR, GFAP (astrocyte marker), and DAPI (nuclear marker), under the indicated conditions. Insets are magnified images of the corresponding boxed regions. Scale bars, 20 μm.