Figure 4
Effects of phthalates on apoptosis-related gene expression in bovine iPSCs and MEFs as feeder cells. (a) Western blotting analysis of the AR-mediated apoptosis-related proteins in cell lysates from iPSCs plus MEFs (left panels) and from MEFs alone (right panels). MEFs were treated with mitomycin C, cultured in the iPSC medium for 2 weeks, and treated with the phthalates indicated (0.1% DMSO-treated control, 10−6 M DEHP, 10−6 M DBP, and 10−6 M BBP) for 24 h, as described in the Materials and Methods, and then harvested. Proteins (30 μg) were loaded into each lane, and each protein was detected using the antibodies indicated. (b) Relative expression values of the blotted proteins in iPSCs and MEF feeder cells. Blots were scanned and quantified using a LI-COR Odyssey near-infrared imaging system. β-Actin (control) was set as 1.0. Intensity of bands in western blotting was quantitated by GeneTools (Syngene) and Image Lab software (Bio-Rad). Relative intensities of each band image in iPSCs were calculated by normalization of corresponding band image of MEFs. (c) Relative mRNA expression levels of AR, p21Cip1, AKT1, AKT2, BAX, and BCL-2 in iPSCs were calculated. The expression level in the control (DMSO treated) was taken as 1.0. Cells were treated with phthalate derivatives (0.1% DMSO control, 10−6 M DEHP, 10−6 M DBP, and 10−6 M BBP). Real-time qPCR was performed using the bovine-specific primers, which were not cross-reacted with mouse, listed in Table 2. Data were expressed as the means±S.D., and a t-test was used to compare them with the results obtained for the DMSO-treated control iPSCs (n≥3, **P<0.01)
