Reporter assay.

HEK293 cells were transfected with the STAT3 reporter plasmid 4×M67 pTATA TK-Luc (57) and nsp5 plasmids. The Renilla luciferase vector pRL-TK (Promega, Madison, WI), which expresses Renilla luciferase under the control of a constitutively active promoter, was also transfected for normalization. At 24 h after transfection, OSM was added into the cells at a final concentration of 10 ng/ml. After incubation for another 24 h, the cells were lysed for the luciferase activity assay of firefly and Renilla luciferases according to the manufacturer's instructions (Promega). Lysates of the cells without OSM treatment were included as a control for the assessment of the STAT3 activation level. The relative firefly luciferase activity is shown after normalization with the Renilla level, as described previously (53).