FIGURE 7.

JAK2 does not mediate RET/PTC3-induced activation of STAT1. PTC3–5 cell line was incubated with Dox (1 μg/ml) or with Dox (1 μg/ml) and JAK2 inhibitor AG490 (5, 10, and 50 μm) for 24 h and harvested for cell lysate preparation or for total RNA extraction and cDNA synthesis. A, immunoblottings for RET, pTyr-905 RET, pSer-727 STAT1, pTyr-701 STAT1, total STAT1, and tubulin were performed. Treatment with AG490 did not appear to influence RET activation and STAT1 phosphorylation and expression. B, IDO1 mRNA expression levels were assayed by quantitative PCR. The data are presented as means of arbitrary units. Inhibition of JAK2 did not significantly influence IDO1 expression. C, the PTC3–5 cell line was transfected with rat JAK2 siRNA (50 nm) (siJAK2) or with non-target siRNA (50 nm) (siNC). Twenty-four hours after transfection, cells were stimulated with Dox (1 μg/ml) for 48 h before cell harvesting for RNA and cell lysate preparation. Immunoblottings for RET, pTyr-701 STAT1, pSer-727 STAT1, total STAT1, JAK2, and tubulin were performed. JAK2 silencing did not appear to influence RET activation and STAT1 phosphorylation and expression. D, IDO1 mRNA expression levels were assayed by quantitative PCR. The data are presented as means of arbitrary units. Silencing of JAK2 did not significantly influence IDO1 expression. All the experiments were repeated twice and the more representative results are depicted.