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. 2008 Jun 4;28(23):5870–5878. doi: 10.1523/JNEUROSCI.5385-07.2008

Figure 4.

Figure 4.

IL8 is a direct STAT3-repressed target gene. A, Diagrammatic representation of the top 50 genes in three independent microarray analyses of control or S3C-expressing U87 cells, ranked according to fold change, that were repressed or induced after S3C expression. B, Top 10 repressed (left) or induced (right) genes after S3C expression. The fold change is indicated, and GenBank numbers are in parenthesis. C, Top, Immunoblotting of lysates of human glioblastoma samples (GBM) with an IL8 antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control [GAPDH immunoblotting of some of the GBM samples is also shown by de la Iglesia et al. (2008)]. Eight of the tumors (#4, 8, 11, 13, 16, 19, 24, and 25) displayed very high levels of IL8. The majority of these tumors (6 of 8) also had low PTEN levels (de la Iglesia et al., 2008). Bottom, Fisher's exact test of IL8-positive and IL8-negative tumors revealed a strong correlation between IL8 expression and low PTEN levels [odds ratio (OR), 14; p < 0.01]. D, Left, Northern analysis of control vector-infected U87 glioblastoma cells or those expressing S3C using an IL8 probe. 18S RNA levels indicate equal loading. Middle, Immunoblotting of lysates of control U87 glioblastoma cells or those expressing S3C or mS3C with an IL8 antibody. The asterisk indicates a nonspecific band. Right, Sandwich ELISA chemiluminescent analysis of medium from the control U87 glioblastoma cells or those expressing S3C using two antibodies specific for human IL8 (R&D Systems). Shown are mean IL8 concentration ± SEM (pg of IL8/mg of cell protein; n = 3; t test, *p < 0.01). E, STAT3 represses the IL8 promoter. Pten−/− astrocytes were transfected with increasing amounts of an expression plasmid encoding S3C or a control plasmid together with an IL8 promoter-controlled luciferase reporter gene and a renilla expression plasmid and subjected to dual luciferase assay 48 h after transfection. Activated STAT3 significantly reduced IL8 promoter activity (n = 3; ANOVA, *p < 0.0001). V, Vector. F, Chromatin immunoprecipitation analysis at the endogenous IL8 promoter in S3C-expressing U87 glioblastoma cells using two different STAT3 antibodies (m, mouse; r, rabbit). Mouse and rabbit HA antibodies were used as controls. Negative controls for the PCR were performed with primers for the E-cadherin gene. STAT3 directly bound to the endogenous IL8 gene.