Cellular system for screening of compounds inhibiting STAT3 transcriptional activity.

(A) Overview of the STAT3 luciferase reporter system. (B) A4wt and A4 sublines were transiently transfected with pGL4.27-SIE reporter and pRL-Renilla and either left untreated or were treated with IL6 + IL6R (50ng/mL and 100 ng/mL respectively) or IFNγ (40 IU/mL). After 6h, luciferase activities were measured using Dual-Glo Luciferase assay. The data presented as ratios to the corresponding untreated control. (C) A4 and A4wt sublines were treated with IL6 + sIL6R (50ng/mL and 100 ng/mL, respectively) or IFNγ (40 IU/mL) for 30 min. Western blotting analysis was performed with the indicated antibodies. GAPDH was used as a loading control. (D) A4wt cells were transfected with pGL4.27-SIE reporter and a stable sub-line A4wt-SIE-6 was selected. Cells were either left untreated (control) or were stimulated with IL6 + sIL6R (50 and 100 ng/mL, respectively) either before treatment with STATTIC (10 μM) or Pyr6 (1 μM), or after, as indicated. Luciferase activity was measured 6h after the first treatment in both cases using SteadyLite^TM Plus. The data is presented as fold induction. Error bars represent SE from quadruplicates. (E) A4wt cells stably transfected with pGL4.27-SIE reporter were seeded in 384-well plates and pre-treated with IL6 and sIL6R (50 and 100 ng/mL, respectively) for 1h, whereafter treated with STATTIC (10 μM) or Pyr6 (1 μM) for 5h to estimate the suitability of the screening system for the high-throughput screening and calculate the z’-factor. Luciferase activity was measured as in (C). One column of the 384-well plate was used for each treatment. The data is presented as average of raw luciferase activity ± standard deviation. The relevant z’-values are described in the text. (F) Outline of the luciferase reporter assay used for high-throughput screening. (G) The dot-blot of the results of the primary screening. A4wt-SIE-6 cells stably transfected with pGL4.27-SIE were pre-treated with IL6 and sIL6R and then treated with the drugs. The data are presented as percent inhibition of luciferase activity where the luciferase activity of the cells treated with IL6+sIL6R+mock is regarded as 0% inhibition (red dots), and the activity in cells treated with IL6+sIL6R+STATTIC regarded as 100% inhibition (green dots). The dashed line is the distance of 3 standard deviations from IL6+sIL6R+mock-treated cells. All the library compounds were used at 10 μM. (H) The STAT3 inhibitor screening funnel.