FIG 7.

NO mediates ASS1 and iNOS induction of STAT3 activation. (A) Depletion of ASS1 expression inhibits STAT3 tyrosine phosphorylation. MM and KMM cells were transduced with 3 ASS1 shRNAs (sh1, sh2, or sh3) or a scrambled shRNA (ctl) and examined for the levels of total and phosphorylated STAT3 (Y705) by Western blotting. The ASS1 protein level was also examined to monitor knockdown efficiencies, while β-tubulin was used as a loading control. (B and C) The NO donor SNAP increases intracellular NO levels. MM and KMM cells were treated with 0.5 mM SNAP for 1 h and examined for intracellular NO by DAR staining. The intracellular NO level was examined with a fluorescence microscope (B), and the relative intensity was quantified with ImageJ (C). (D) ASS1 knockdown has no effect on ROS production. MM and KMM cells transduced with 3 ASS1 shRNAs (sh1, sh2, or sh3) or a scrambled shRNA (ctl) for 2 days were examined for intracellular ROS levels. (E) The NO donor SNAP partially rescues STAT3 activation following ASS1 knockdown. MM and KMM cells transduced with an ASS1 shRNA (shRNA3) or a scrambled shRNA (ctl) for 2 days were treated with 0.5 mM SNAP for 0.5 h and then examined for the levels of total and phosphorylated STAT3 (Y705) by Western blotting. β-Tubulin was used as a loading control. APC-A indicates allophycocyanin (APC) fluorescent intensity.