The androgen receptor (AR) has a critical role in promoting androgen-dependent and -independent apoptosis in testicular cells. ratio and large nucleoli.15 The estimated reprogramming efficiency of our one-factor method was 0.3% which is 20-fold Apremilast higher than that of the one-factor approach used for reprogramming murine neural stem cells.16 The cells exhibited a strong alkaline phosphatase activity after we continued the culture for >4 weeks (Figure 1a). Immunofluorescence staining confirmed that this iPSCs induced by (1F-iPSCs) expressed stemness markers such as OCT4 NANOG SOX2 SSEA-1 and SSEA-4 (Physique 1a). These markers were more intense in the dense patches of cells. Reverse transcription-PCR (RT-PCR) analysis confirmed the expression of ESC markers in 1F-iPSCs including (Physique 1b). A cytogenetic study based on G-banding exhibited normal distributions of the 60 chromosomes in the iPSCs including the XY sex chromosomes at passage 15 (Physique 1c). Physique 1 Generation of iPSCs from bovine testicular cells. (a) Common morphology of bovine iPSC colonies generated using on day 25 after electroporation Apremilast ( × 100 magnification; upper left panel). Alkaline phosphatase staining of bovine iPSCs (lower … Pluripotency To confirm the developmental potential of the bovine 1F-iPSCs differentiation of and marker expression by bovine iPSC-derived ectodermal mesodermal and endodermal precursor cells. Immunostaining with antibodies directed against the astrocyte-specific antigen GFAP (ectodermal … Effects of phthalate esters Next we examined cytotoxicity necrosis and apoptosis in the bovine testicular cells and iPSCs generated from the same testicular cells following exposure to DEHP DBP and BBP. The three phthalates induced significant cytotoxicity in iPSCs compared with the original testicular cells even at low concentrations (10?6 to 10?8?M; Supplementary Figure S1A). Interestingly the phthalates induced a higher level of necrosis in the testicular cells compared with the iPSCs (Supplementary Figure S1B) Apremilast whereas the phthalate esters elicited significant apoptotic activity in the iPSCs which we evaluated using annexin V staining (about 2.2-3.3-fold; Figure 3a). This was also supported by the observations of a higher caspase 3 activity (about 4.5-6.8-fold; Figure 3b) and an increased sub-G1 cell population (about 5.2-8.4-fold; Supplementary Figure S1C) in the phthalate ester-treated iPSCs. These results suggest that the phthalate esters (DEHP DBP and BBP) induced apoptosis in bovine testicular cell-derived iPSCs. Figure 3 Apoptosis induced by phthalate derivatives in bovine iPSCs. (a) Fluorescein isothiocyanate-labeled annexin V staining followed by flow cytometry to identify apoptotic cells as described TNF in the Materials and Methods. DEHP DBP or BBP were added at doses … Screening specific antibodies for proteins from bovine iPSCs using a microwestern array (MWA) To understand the signaling involved with apoptosis in testicular iPSCs exposed to phthalate esters we used a MWA 17 which facilitated the high-throughput assessment of protein abundance after the electrophoretic separation of 96-well microarray cell lysates. We screened a series of antibodies to identify appropriate antibodies which detected bovine and mouse proteins (Supplementary Figure S2A). To maintain the characteristic stemness of iPSCs they had to be cultured with mitomycin C-treated MEF as feeder cells. Without the feeder cells the stemness features were lost rapidly based on staining Apremilast for alkaline phosphatase and SSEA 1 or 4 (data not shown). Thus we had to examine samples from iPSCs with MEF and from MEF alone to compare the relative expression levels of apoptosis-related proteins (Supplementary Figure S2B). The results suggested that the protein levels of BAX and p21Cip1 (cycling-dependent kinase inhibitor 1) were increased in phthalate-treated iPSCs which were normalized against the levels in MEF feeder cells. Increased BAX/BCL-2 ratio in phthalate ester-treated bovine testicular iPSCs Next we conducted traditional western blot analyses to verify the results obtained by MWA. Samples from iPSCs with MEF feeder cells and from MEF feeder cells alone were prepared as described above. We found that the expression degree of the proapoptosis Apremilast proteins BAX was improved in iPSCs by treatment with DEHP DBP and BBP (about 2.6-3.0-fold Figures 4a and b) following normalizing against the expression levels in MEF feeder cells. By.