FIGURE 6.
STAT1 Ser-727 phosphorylation depends on activation of the MAPK and PI3K pathways. A, BRAF9.6 cell line was incubated with Dox (1 μg/ml) for the indicated times and harvested for cell lysate preparation. Immunoblottings for pERK, IDO1, pTyr-701 STAT1, pSer-727 STAT1, total STAT1, and tubulin were performed. BRAFV600E expression only induced phosphorylation of serine 727 without affecting tyrosine 701 phosphorylation and STAT1 and IDO1 expression levels (PC: positive control). B, PTC3–5 cells were incubated with Dox (1 μg/ml) for the indicated times in the presence or absence of the MEK inhibitor U0126 (10 μm) and harvested for cell lysate preparation. Immunoblottings for RET, pTyr-701 STAT1, pSer-727 STAT1, total STAT1, pERK, and tubulin were performed. Inhibition of RET/PTC3-induced activation of the MAPK pathway appeared to strongly inhibit STAT1 serine 727 phosphorylation and, to a lower extent, STAT1 tyrosine 701 phosphorylation, without affecting the expression levels of the transcription factor. C, PTC3–5 cells were incubated with Dox (1 μg/ml) for the indicated times in the presence or absence of the PI3K inhibitor wortmannin (0.3 μm) and harvested for cell lysate preparation. Immunoblottings for RET, pTyr-701 STAT1, pSer-727 STAT1, total STAT1, pAKT, and tubulin were performed. Inhibition of RET/PTC3-induced activation of the PI3K pathway appeared to inhibit significantly STAT1 serine 727 phosphorylation and, to a lower extent, STAT1 tyrosine 701 phosphorylation, without affecting the expression levels of the transcription factor. D, the STAT1 pTyr-701 and pSer-727 bands of three independent experiments were analyzed by densitometry and normalized toward tubulin. The results indicate that either the MAPK or PI3K pathway inhibition cause a not significant light reduction of STAT1 tyrosine 701 phosphorylation, but a statistically significant strong reduction of STAT1 serine 727 phosphorylation. E, PTC3–5 cells were incubated with Dox (1 μg/ml) for the indicated times in the presence or absence of the MEK inhibitor U0126 (10 μm) and harvested for total RNA preparation. After total RNA extraction and cDNA synthesis, IDO1 mRNA expression levels were assayed by quantitative PCR. The data are presented as means of arbitrary units. Inhibition of RET/PTC3-induced activation of the MAPK pathway appeared to strongly inhibit IDO1 mRNA expression (*, p = 0.0022). F, PTC3–5 cells were incubated with Dox (1 μg/ml) for the indicated times in the presence or absence of the PI3K inhibitor wortmannin (0,3 μm) and harvested for total RNA preparation. After total RNA extraction and cDNA synthesis, IDO1 mRNA expression levels were assayed by quantitative PCR. The data are presented as means of arbitrary units. Inhibition of RET/PTC3-induced activation of the PI3K pathway appeared to strongly inhibit IDO1 mRNA expression (*, p = 0.01). All experiments were repeated twice and the more representative results are depicted. p values were calculated applying the two-tailed unpaired Student's t test.