Sun et al. Journal of Experimental & Clinical Cancer Research 2011, 30:82 http://www.jeccr.com/content/30/1/82 Page 4 of 10 FACSAria flow cytometer (Becton Dickinson). The ratio of Tregs to CD3+T cells before culture was also assessed. The data were analyzed using Cell Quest soft- ware (Becton Dickinson). Statistical Analysis All data were expressed as (x¯ ± SD) and analyzed with statistical package SPSS 11.5 for Windows (SPSS Inc., Chicago, IL). The SNK-q method was used to determine statistically significant differences among the groups. One-way analysis of variance (ANOVA) and the Stu- dent’s t test were used to determine the means of two different groups. P < 0.05 was considered statistically significant. Results Identification of the recombinant plasmid pIRES2-EGFP- IDO Digestion of the pIRES2-EGFP-IDO construct with BglII and SalI liberated an IDO insert of the expected length (1225 kb), indicating that the plasmid was successfully constructed (Figure 1A). Analysis of IDO expression by PCR using genomic DNA, or by RT-PCR using total RNA, yielded a 188 bp fragment; meanwhile, no IDO expression was detected in CHO/EGFP cells, indicating that we could specifically detect the integration into the CHO cell genome and transcription of the transfected IDO gene (Figure 1B). Western blot analysis showed that the stably transfected IDO+ CHO cells expressed the 42 kDa IDO protein (Figure 1C). Kynurenine (8.14 ± 1.02 mg/L) but not tryptophan (< 3 pmol) was detected in the culture supernatant 72 h after the CHO cells were incubated with the IDO construct. However, tryptophan (5.85 ± 0.74 mg/L) but not kynurenine was detected in the culture supernatant of CHO/EGFP cells, indicating that IDO expressed by transfected CHO cells possessed functional activity and could metabolize tryp- tophan (Figure 1D). Effect of IDO+ CHO cells on CD3+T cell apoptosis After 72 h of co-culture of CD3+T cells and IDO+ CHO cells, 79.07 ± 8.13% of CD3+T cells were Figure 1 Identification of IDO transfected CHO cells. (A) Identification of recombinant plasmid pIRES2-EGFP-IDO by restriction enzyme analysis. The plasmid pIRES2-EGFP-IDO can be digested with BglIIand SalI. xperiments in this figure and following figures were performed at least three times on separate occasions. (B) IDO gene integration and transcription by PCR and RT-PCR. (C) Western blot analysis of IDO protein expression in CHO-IDO cells using anti-IDO antibody. In transfected group, CHO cells transfected with IDO expressed the 42 kDa IDO protein, indicating that CHO cells stably transfected with IDO could produce IDO protein. (D) Analysis of free amino acids in culture supernatant. Amino acid level in CHO cells 72 h after IDO transfection: (His) 33.75 mg/L, (Kyn) 7.03 mg/L, (Trp) < 3 pmol. Amino acid level in CHO cells with pIRES2- EGFP transfection 72 h after culturing: (His) 38.12 mg/L, (Trp) 5.63 mg/L, (Kyn) < 3 pmol. His: histidine; Trp: trytophan; Kyn: kynurenine.