STAT1C significantly decreases STAT3 transcriptional activity. (A) STAT3 luciferase reporter assay showed that STAT1C significantly decreased STAT3 transcriptional activity in a doxycycline dose-dependent manner. Treatment of doxycycline (200 ng/mL) in Tet on-SupM2 EV cells showed that low-dose doxycycline has no appreciable effects on STAT3 transcriptional activity. (B) STAT3 consensus DNA probe binding assay showed that STAT1C sequestered STAT3 DNA binding ability in a doxycycline dose-dependent manner, and the western blots in the right panel showed the input of cell nuclear lysates. Histone deacetylase 1 (HDAC-1) was used as loading control for nuclear lysates. (C) Co-immunoprecipitation assay showed that STAT1C induced more STAT1/STAT3 heterodimer formations in a doxycycline dose-dependent manner at the expense of STAT3 homodimers. The right panel shows the input. Statistical significance was calculated by Student t test. *P < .05; **P < .01. IB, immunoblotting.