Skip to main content
. 2012 Jun 6;32(23):7806–7818. doi: 10.1523/JNEUROSCI.3243-11.2012

Figure 2.

Figure 2.

STAT3 directly regulates iNOS transcription in astrocytes. A, EGFRvIII;Stat3loxP/loxP and EGFRvIII;Stat3−/− astrocytes transfected with a luciferase reporter plasmid driven by a promoter containing the 2 or 0.3 kb region upstream of the iNOS transcriptional start site or the control pGL2-basic reporter plasmid together with a Renilla expression plasmid were subjected to dual luciferase assay 48 h after transfection. Expression of the luciferase reporter from both the 2 and 0.3 kb regions of the iNOS promoter was significantly increased in EGFRvIII;Stat3loxP/loxP astrocytes compared with EGFRvIII;Stat3−/− astrocytes (Kruskal–Wallis test, p < 0.01, n = 4). B, EGFRvIII;Stat3loxP/loxP and EGFRvIII;Stat3−/− astrocytes transfected with the 0.3 kb iNOS–luciferase reporter plasmid together with dominant-negative Stat3 (STAT3D) or the control vector and the Renilla expression plasmid were subjected to dual luciferase assay 48 h after transfection. Expression of the iNOS–luciferase reporter was significantly increased in EGFRvIII;Stat3loxP/loxP astrocytes compared with EGFRvIII;Stat3−/− astrocytes in the background of control vector. Expression of STAT3D significantly reduced the expression of the iNOS–luciferase reporter in EGFRvIII;Stat3loxP/loxP astrocytes (Kruskal–Wallis test, p < 0.005, n = 4). C, EGFRvIII;Stat3loxP/loxP and EGFRvIII;Stat3−/− astrocytes transfected with vector control, STAT3D, or STAT3C were subjected to immunocytochemistry using the GFP and iNOS antibodies. Endogenous iNOS expression was substantially reduced in EGFRvIII;Stat3loxP/loxP astrocytes upon expression of STAT3D and substantially increased in EGFRvIII;Stat3loxP/loxP and EGFRvIII;Stat3−/− astrocytes upon expression of STAT3C. Scale bar, 20 μm. D, Lysates of EGFRvIII-expressing and control MSCV-infected astrocytes transfected with wild-type Stat3 (STAT3 WT), STAT3D, or STAT3C were immunoblotted with the phospho-Tyr (4G10), EGFR, STAT3, or actin antibody. Expression of STAT3D or STAT3C had little or no effect on the tyrosine phosphorylation of EGFRvIII. E, Top, STAT3 binding site in iNOS 94 bp upstream of the transcriptional start site (TSS) is conserved. Bottom, Mutations introduced into the iNOS promoter to disrupt STAT3 binding. F, EGFRvIII;Stat3loxP/loxP and EGFRvIII;Stat3−/− astrocytes transfected with the 0.3 kb iNOS–luciferase or 0.3 kb iNOS–luciferase mutant reporter plasmid together with the Renilla expression plasmid were subjected to dual luciferase assay 48 h after transfection. Expression of the iNOS–luciferase reporter was significantly increased in EGFRvIII;Stat3loxP/loxP astrocytes compared with EGFRvIII;Stat3−/− astrocytes (Kruskal–Wallis test, p < 0.005, n = 5). Mutation of the STAT3 binding site significantly reduced iNOS-promoter-mediated expression in EGFRvIII;Stat3loxP/loxP astrocytes (Kruskal–Wallis test, p < 0.005, n = 5). G, EGFRvIII;Stat3loxP/loxP astrocytes transfected with the 0.3 kb iNOS–luciferase or 0.3 kb iNOS–luciferase mutant reporter plasmid together with STAT3C or the control vector and the Renilla expression plasmid were subjected to dual-luciferase assay 48 h after transfection. Expression of STAT3C significantly increased expression of the iNOS–luciferase reporter (Kruskal–Wallis test, p < 0.0005, n = 5) but had little or no effect on the expression of the iNOS–luciferase mutant reporter. H, EGFRvIII;Stat3loxP/loxP and EGFRvIII;Stat3−/− astrocytes were subjected to ChIP analyses using the STAT3 antibody. The STAT3 gene served as positive control. Endogenous STAT3 was significantly enriched at the endogenous iNOS promoter in EGFRvIII;Stat3loxP/loxP astrocytes compared with EGFRvIII;Stat3−/− astrocytes (ANOVA, p < 0.001, n = 3).