Effect of selected HAK compounds on OSM-induced STAT3 phosphorylation at serine 727 in U343 cells. Treatment of U343 cells with HAK compounds led to a significant reduction of OSM-induced pSTAT3S727, while STAT3 expression was not affected. For western blot analysis (A) 0.25 × 106 cells co-treated for 6 h with OSM (100 ng/ml) and selected HAK compounds (20 μM) were extracted and analyzed as described in experimental procedures. Blots (30 μg protein/lane) were probed with STAT3 and pSTAT3S727-specific antibodies (1:500, overnight). For immunocytochemistry (B) 0.1 × 106 cells were grown on cover slips and treated identically as described above. Post treatment, cells were fixed in methanol and labeled with a pSTAT3S727-specific antibody (1:200, overnight). From each sample 15 images were taken by means of fluorescence microscope and app. 250 cells/sample were densitometrically analyzed by MetaMorph imaging software (C). Mean signal intensity of OSM-treated control cells was set to 100%. All data are presented as mean ± SD from experiments carried out in triplicate. ns, non significant; ***p < 0.001.