Changes in TGF-β1 and TGF-β2 expression in a coculture model.

<p>(A) TGF-β1 and TGF-β2 mRNA levels in GC cells after direct cocultures were increased compared to monoculture, but there were no significant differences in TGF-β1 and TGF-β2 mRNA levels in GC cells, irrespective of the origin of the PBMCs (GC patients or controls). (B) TGF-β1concentrations in the cell supernatant of cocultures were significantly increased compared to those in PBMCs or GCs cultured alone in a FBS-free environment (<i>P</i><0.05). Its levels in the direct coculture group were significantly higher than those in the indirect group (<i>P = </i>0.029). TGF-β2 levels were also increased in direct cocultures, but the differences after cocultures were not significant. (C) Cytokine production levels were significantly increased in indirect coculture groups after the addition of FBS (<i>P</i><0.05), but no obvious change was detected in direct coculture ones. The experiment was conducted twice. All data are shown as means ± SD of triplicates. (D) Origin of cytokines. In GC cells, TGF-β1 mRNA levels were increased approximately 3-fold in the direct coculture and increased 2-fold in the indirect one compared to monocultures; TGF-β2 mRNA levels were significantly increased after direct coculture but not statistically changed after indirect one. In PBMCs, TGF-β1 mRNA levels were significantly decreased and TGF-β2 levels were remarkably increased after cocultures. Levels were normalized to GAPDH, and levels in the monoculture group were defined as 1.0. All data are shown as means ± SD. (E) The mRNA levels of Smad2 and Smad3 in GC cells were significantly increased after cocultures (<i>P</i><0.05), which were higher in the direct coculture than those in the indirect one, but there was no statistic difference in the levels of Smad4. (F) Cell-IQ showed that the addition of exogenous TGF-β1 (25 ng/mL) to GC cells suppressed the growth and division of tumor cells, but with no significant difference. Eight images from different visual field were analyzed for each group. (G) Cell Counting Kit-8 (CCK-8) assay showed that TGF-β1 (25 ng/mL) inhibited the viability of PBMCs significantly at 72 h. The line shows the inhibition ratio of TGF-β1 stimulated cells compared to untreated controls. <i>GC:</i> gastric cancer; <i>PMBC:</i> peripheral blood mononuclear cell; <i>Dir-co:</i> direct coculture; <i>Ind-co:</i> indirect coculture; <i>Mono:</i> monoculture; <i>FBS:</i> foetal bovine serum. <i>ns,</i> not significant; *, <i>P</i><0.05; **, <i>P</i><0.05.