FIGURE 3. The iUC-MSC lines exhibit higher proliferation capability, which can be effectively reversed by FLP recombinase.

(A) Cell proliferation assessed by crystal violet staining. Exponentially proliferating primary UC86 cells (a) and iUC86 cells (b) were seeded at the same density and subjected to crystal violet staining at the indicated time points. Representative results are shown. (B) & (C) Cell proliferation and viability assays. Exponentially proliferating primary UC86 cells (a) and iUC86 cells (b) were seeded at the same density and subjected to MTT assay (B) or counting for viable cells after Trypan blue staining (C). “*” p<0.05; “**” p<0.001, compared with that of the primary cell groups. (D) & (E) Decreased cell proliferation caused by FLP-mediated removal of SV40 T antigen. Subconfluent iUC86 and iUC79 (not shown in C) cells were infected with Ad-GFP (a) or Ad-FLP (b) and subjected to crystal violet staining at the indicated time points. Representative results are shown. The stained cells were dissolved and subjected to OD_590nm reading. “*” p<0.05; “**” p<0.001, compared with that of the GFP groups. (F) FLP-mediated removal of SV40 T antigen in the iUC-MSC lines. Subconfluent iUC86 and iUC79 cells were infected with Ad-GFP or Ad-FLP and subjected to crystal violet staining at the indicated time points. Representative results are shown. The stained cells were dissolved and subjected to OD_590nm reading. “*” p<0.05; “**” p<0.001, compared with that of the GFP groups. (F) FLP-mediated removal of SV40 T antigen in the iUC-MSC lines. Subconfluent iUC86 and iUC79 cells were infected with Ad-GFP or Ad-FLP for 72h. Total RNA was isolated and subjected to reverse transcription and semi-quantitative PCR using primers specific for SV40 T antigen. GAPDH was used as a reference control. PCR products were resolved on 1.5% agarose gels. PCR reactions were done in triplicate and representative results are shown.