Figure 7.

TGF-β produced by TLR4 expressing cells upon stimulation with HMGB1 triggers galectin-9 expression in target cancer cells. Active TLR4 is capable of inducing the same effects without HMGB1. (A) THP-1 cells were treated with 1 mM H₂O₂ for 24 h and the medium was collected for further use. This medium after H₂O₂ degradation with or without immunoprecipitation of HMGB1 was used to treat PMA-differentiated THP-1 cells expressing high levels of TLR4 for 24 h. The medium from PMA-treated THP-1 cells treated with HMGB1-containing medium was collected. This medium with or without immunoprecipitation of TGF-β was used to incubate MCF-7 cells for 24 h followed by measurement of galectin-9 by Western blot. (B) Colo 205 cells were transfected with constitutively active TLR4 or corresponding empty vector followed by 24h of incubation without further treatment. Expression of Tim-3 protein and it’s secretion (values are presented in the text), galectin-9 protein expression by both Western blot and ELISA and secretion by ELISA were analysed. (C) TGF-β secretion and Smad3 activating phosphorylation were also detected. Images are from one experiment representative of 4 giving similar results. Quantitative data are shown as mean values ± SEM from 4 independent experiments. *p < 0.05 and **p < 0.01 vs control.