Figure 3

FLLL32 decreased STAT3 DNA binding in OSA cell lines. A) Canine OSA cell line OSA8 and human OSA cell line SJSA were incubated with media, DMSO, 10 μM curcumin, or 10 μM FLLL32 for 4 hours. Cells were harvested and nuclear protein isolated. Nuclear protein was added to binding reactions with labeled species specific DNA probes for the STAT3 recognition sequences located in the promoter for survivin in the presence or absence of unlabelled competitor probe. Additionally, anti-STAT3 antibody was added to nuclear protein from cells treated with media alone to demonstrate specificity of the binding reaction. Reactions were separated on an acrylamide gel, transferred to a nylon membrane, and the DNA was crosslinked. The membranes were processed using the LightShift Chemiluminescent EMSA kit (Thermo Fisher Scientific Inc, Rockford, IL). Experiments were repeated two times. B) Canine OSA cell line OSA8 and human OSA cell line SJSA were incubated with media, DMSO, 10 μM curcumin, or 10 μM FLLL32 for 4 hours concurrently with cells treated for EMSA above. Cells were harvested and total protein isolated and quantified. Protein was separated by SDS-PAGE. Western blotting was performed for STAT3 and β-actin. Experiments were repeated two times.