Sun et al. Journal of Experimental & Clinical Cancer Research 2011, 30:82 http://www.jeccr.com/content/30/1/82 Page 8 of 10 Figure 4 Foxp3 expression in T cells after co-culture was detected by RT-PCR, Real-time PCR or Western blot. (A) Analysis of RT-PCR products of Foxp3 and comparison of the gray scale value between Foxp3 and b-actin by agarose gel electrophoresis. Three separate experiments were carried out. RT-PCR product of b-actin and Foxp3 from the total mRNA isolated from CD3+T cells cultured with growth medium, or from the T cells co-cultured with IDO gene-transfected CHO cells, or from the T cells co-cultured with CHO/EGFP cells. The value of the Foxp3 and b-actin gray scale ratio in T cells after 7 days of co-culture with IDO gene-transfected CHO cells was higher than in the control groups (P < 0.05). (B) Expression of Foxp3 gene analyzed by real-time RT-PCR. Three separate experiments were carried out. Amplification curve of Foxp3 in the IDO gene-transfected group and the control groups. Expression of Foxp3 in T cells after 7 days of co-culture with IDO gene- transfected CHO cells was higher than that in the control groups (P < 0.05). (C) Expression of Foxp3 analyzed by Western blot analysis. Three separate experiments were carried out. Expression of Foxp3 protein in the CD3+T cells cultured with growth medium for 7 days; or 7 days after co-culture with CHO/EGFP cells; or 7 days after co-culture with IDO+ CHO cells. No Foxp3 protein was detected in the control groups. cells in the lymph nodes draining tumors that express IDO had local infiltration of Tregs cells [21,22,22,24]. Furthermore, when IDO was expressed in the primary tumor of breast cancer patients, there was a direct cor- relation between an increase in volume of the primary breast cancer tumor and the proportion of Tregs in the peripheral circulation [23]. Tregs cells are also likely to be involved in IDO-mediated tumor immune tolerance [11,12]. To investigate this hypothesis, we established a CHO cell line that stably expressed IDO. Western blot analysis confirmed that CHO cells trans- fected with IDO expressed IDO protein with an expected molecular weight of approximately 42 kDa. At the same time, we detected a decrease in trypto- phan in the culture medium, and an increase in its metabolite kynurenine, suggesting that IDO expressed by the transfected cells was functional and could lead to the depletion of tryptophan in the environment.