Figure 1. STAT3 regulates iNOS expression in EGFRvIII-expressing astrocytes.

(A) RNA was isolated from mouse EGFRvIII;Stat3^loxP/loxP, EGFRvIII;Stat3^−/−, PTENi;Stat3^loxP/loxP, and PTENi;Stat3^−/− astrocytes and subjected to RT-PCR with primers specific for the indicated genes. GAPDH served as control. iNOS mRNA levels were specifically reduced in EGFRvIII;Stat3^−/− astrocytes compared to EGFRvIII;Stat3^loxP/loxP astrocytes.

(B) RNA isolated from mouse EGFRvIII;Stat3^loxP/loxP and EGFRvIII;Stat3^−/− astrocytes was subjected to quantitative RT-PCR analyses using primers specific for STAT3 and iNOS. mRNA levels were normalized to GAPDH. STAT3 and iNOS mRNA levels were significantly reduced in EGFRvIII;Stat3^−/− astrocytes compared to EGFRvIII;Stat3^loxP/loxP astrocytes. (ANOVA, p < 0.0005, n = 3).

(C) EGFRvIII;Stat3^loxP/loxP and EGFRvIII;Stat3^−/− astrocytes were subjected to immunocytochemistry using the rabbit iNOS antibody. Representative images are shown. The expression of iNOS was substantially reduced in EGFRvIII;Stat3^−/− astrocytes compared to EGFRvIII;Stat3^loxP/loxP astrocytes. Scale bar = 20 μm.

(D) Lysates of EGFRvIII;Stat3^loxP/loxP and EGFRvIII;Stat3^−/− astrocytes were immunoblotted with the iNOS or α-tubulin antibody. The levels of iNOS protein were substantially reduced in EGFRvIII;Stat3^−/− astrocytes compared to EGFRvIII;Stat3^loxP/loxP astrocytes.

(E) RNA isolated from mouse PTENi;Stat3^loxP/loxP and PTENi;Stat3^−/− astrocytes was subjected to quantitative RT-PCR analyses using primers specific for iNOS. mRNA levels were normalized to GAPDH. iNOS mRNA levels were not significantly different between PTENi;Stat3^loxP/loxP astrocytes compared to PTENi;Stat3^−/− astrocytes.