Fig. 4. FL3 reduces IL-6-induced STAT3 transcriptional activity and inhibits induction of HIF1α.

A Luciferase assay of HuH7 and HLF cells measuring endogenous STAT3 transcriptional activity using STAT3 binding elements upon treatment with 20 ng/mL IL-6 in the absence or presence of FL3 (100 nM) for 6 h. Renilla luciferase was used as an internal transfection control and for normalization. Data were normalized to corresponding IL-6 stimulated control cells. *p < 0.05. B To test the effect of SH2D4A on STAT3 transcriptional activity, HuH7 cells infected with pTRIPZ-SH2D4A or pTRIPZ-ALB as control and treated with doxycycline (DOX) 1 day after transfection of luciferase and Renilla reporter constructs. Luciferase activity upon treatment with 20 ng/mL IL-6 in the absence or presence of FL3 (100 nM) for 6 h was measured. Data are represented as mean ± SD of three independent experiments with each data point representing the mean of two technical replicates within each experiment. *p < 0.05 for pairwise comparison and ANOVA test was performed on the three mean values of independent experiments comparing all four groups. C Western blots of HuH7 and HLF cells upon stimulation with 20 ng/ml IL-6 or/and FL3 (100 nM) for 15 min or 6 h, as indicated. D FL3 reduced DMOG-induced HIF1α protein level dose-dependently. Cells were stimulated with 1 µM DMOG and FL3 in two different concentrations (50 nM and 100 nM) for 4 h.