Figure 2.

CYD0618 directly binds to STAT3 oncoprotein. A, chemical structure of biotinylated CYD0618 (biotin–CYD0618). B, biotinylation modification does not attenuate the growth inhibition of CYD0618 in A2780 cells. The A2780 cell lysates were incubated with biotin or biotin–CYD0618 in the absence or presence of unlabeled CYD0618 (10-fold) at 4 °C overnight, followed by pulldown with streptavidin–agarose beads. The precipitates were then resolved by SDS-PAGE, followed by silver staining. The indicated bands were excised and analyzed by MS (B) or detected by Western blotting for STAT3 protein as indicated (C). D, CETSA using A2780 cell lysates, which were exposed to CYD0618 (20 μm) or DMSO. Statistical results from four independent experiments are shown. E, recombinant His–STAT3 was incubated with biotin–CYD0618 in the absence or presence of excess unlabeled CYD0618 or CYD–NC for 1 h at 37 °C. IB, immunoblot. F, recombinant His–STAT3 was incubated with biotin–CYD0618 for the indicated times at 37 °C. After streptavidin–agarose pulldown, the mixtures were blotted for His antibody. Data are expressed as mean ± S.D., n = 4. *, p < 0.05 versus vehicle control.