The tyrphostin AG490 inhibits constitutive activation of STAT3 and proliferation of the HD cell line L428.
(A) Inhibition of STAT3-DNA binding by the tyrphostin AG490. L428 cells were cultured as described in “Materials and methods.” The cells were incubated in 0.1% DMSO (control, nuclear extract isolated after 24 hours) or 50 μM AG490 (nuclear extract isolated after 6 and 24 hours). Five micrograms of protein of each probe was analyzed in a gel retardation experiment using radiolabeled oligonucleotides containing the consensus STAT1/3 binding sites as described in the legend to Figure 1. The positions of the STAT3/3 homodimer and the STAT1/3 heterodimer are indicated by arrows. (B) Western blot analysis of phosphorylated STAT3. Nuclear extracts (20 μg) of each cell line were analyzed in a 10% SDS-polyacrylamide gel, transferred to a nylon membrane, incubated with an antibody specific for phosphorylated STAT3, and visualized by ECL. (C) Western blot analysis of unphosphorylated STAT3. The same nylon membrane as in panel B was stripped and reprobed with an antibody specific for STAT3 and the protein was visualized by ECL. (D) Inhibition of proliferation of L428 cells by the tyrphostin AG490. The cells were incubated for 3 days in 0.1% DMSO (control) or 50 μM or 100 μM AG490. Sixteen hours before harvest 0.4 μCi/well3H-thymidine was added to the cells. The results are shown as relative inhibition of proliferation. The proliferation of L428 cells was not affected by DMSO.
