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. 2012 Feb 1;32(5):1826–1846. doi: 10.1523/JNEUROSCI.3380-11.2012

Figure 1.

Figure 1.

Hes-1 suppresses GluR1 promoter activity and GluR1 expression in primary cortical neurons. A, Schematic diagram of the 2 kb rat GluR1 genomic DNA fragment consisting seven putative N-boxes (CACNAG or its reversed form CTNGTG). −1975 is relative to the translational start site (+1). B, The Flag-Hes-1WT plasmid, its dominant-negative form Hes-1-43A44A47A, or Flag vector alone, and GluR1 promoter-luciferase plasmid (pGL3-GluR1-P) were cotransfected to primary cortical neurons, and GluR1 promoter activity was determined 48 h later. C, The Flag-Hes-1WT plasmid, Flag-Hes-5WT plasmid or Flag vector alone (0.4 μg each), and GluR1 promoter-luciferase plasmid (pGL3-GluR1-P) (0.6 μg) were cotransfected to primary cortical neurons, and GluR1 promoter activity was determined 48 h later. Renilla luciferase as used as an internal control. D, Different doses of Hes-1WT plasmid (0.1, 0.2, 0.4, 0.8 μg) and GluR1 promoter construct were cotransfected to primary cortical neurons, and GluR1 reporter assay was performed 48 h later. E, Hes-1 siRNA (40 nm) or control siRNA was cotransfected with GluR1 promoter construct to primary cortical neurons and GluR1 reporter assay was performed 48 h later. Effectiveness of Hes-1 siRNA transfection was confirmed by Western blot. The Renilla signal was used as an internal control. Data are expressed as mean ± SEM. Experiments are in duplicate or triplicate. F, Immunohistochemistry showing the colocalization of NeuN and Hes-1 as well as NeuN and GluR1 in primary cortical neurons. DAPI was used for nucleus staining. Scale bars: top, 15 μm; bottom, 20 μm. G, Western blot showing the nuclear and cytosol expression of JNK1 and Hes-1 in adult rat brain. H, Flag-Hes-1WT plasmid and GFP plasmid were cotransfected to primary cortical neurons at DIV6 and immunofluorescence staining for Hes-1 (red), GluR1 (red), and GFP (green) was performed on DIV8. Quantified results are shown on the right. Scale bar, 10 μm. I, Hes-1 siRNA was cotransfected with GFP plasmid on DIV6, and immunofluorescence staining against Hes-1 (red), GluR1 (red), and GFP (green) was examined on DIV8. Quantified results are shown on the right. GFP transfection and staining was performed for identification of the transfected neuron. Scale bars: top, 20 μm; bottom, 10 μm. The number of cells ranged from 15 to 20 for each group. Data are expressed as mean ± SEM. #p < 0.001.