Figure 4

Glucosamine inhibits STAT3 activity in DU145 cells. A, Control and glucosamine-treated cells were grown in 6-well plates either without or with 2 mM glucosamine and whole-cell extracts were analyzed by Western blot 2, 4, 8 and 24 h after glucosamine treatment. B, EMSA. Lane 1, free probe without nuclear proteins. Lanes 2-5, binding of the STAT3 radioactive probe to nuclear proteins from DU145 cells, untreated (lane 2) or treated with 2 mM glucosamine for 1 h (lane 3), 4 h (lane 4) and 8 h (lane 5). Lane 6-7, competition assay with 100-fold molar excess of non-radioactive STAT3-specific competitor (lane 6) or nonspecific competitor (lane 7). Lane 8, nuclear proteins from untreated DU145 cells incubated with STAT3-specific antibody before incubation with radioactive probe. Representative data from three independent experiments with similar results is shown. C, cells were transfected with pSTAT3-Luc luciferase reporter plasmid under the control of STAT3 responsible element, or control pTALuc plasmid, and were cultured without or with 1 mM glucosamine for 48 hours before luciferase activities were measured in cell extracts. The results are presented as mean of three independent experiments ± standard deviation.