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All experiments were plated in sextuplicate and were performed three times. Flow cytometry was performed as previously described (Kim et?al. 2007). In brief, P19CL6 cells, and cells stably transfected with GATA-6 or PPAR�� expression vector were seeded at 5?��?105 cells per well in a 6-well plate. After 24?h, the cells were administered 10, 50, and 100?nmol/L insulin, and the cells were harvested at 24?h and fixed with 70% ice-cold ethanol overnight at ?20��C. Next, the cells were stained with propidium iodide (PI; Sigma) solution for 30?min at room temperature in the dark. Flow cytometric INPP5D analysis was performed using a BD FACScan flow cytometer (Becton Dickinson Immunocytometry System), and cell cycle were analyzed using Cell-Quest software (BD Biosciences). The experiments were performed three times in duplicate. Undifferentiated P19CL6 cells (day 0), and day 18 of cardiac differentiation P19CL6 cells and/or treated with 10, 50, 100?nmol/L insulin at day 0 of cardiac differentiation for 24?h were dispersed into individual cells with trypsin, dispersed cells were washed with PBS two times and fixed by using 4% formaldehyde for 10?min at 37��C. Fixed cells were incubated in PBS with 0.1% Triton X-100 for 15?min at room temperature, followed by a centrifugation (300?g for 5?min). Next, the cells were incubated overnight at 4��C in PBS containing 0.1% Triton X-100 and MF-20 (1:100), followed by secondary fluorescein isothiocyanate AZD4547 in vivo (FITC)-conjugated goat anti-mouse IgG antibody (Sigma, 1:100) for 1?h at room temperature. Cells were then washed, resuspend in 0.5?mL PBS, and analyzed selleck products using a BD FACScan flow cytometer (Becton Dickinson Immunocytometry System). P19CL6 cells, and cells stably transfected with GATA-6 or PPAR�� expression vector were allowed to adhere to glass coverslips (22?��?22?mm) in 6-well plates. Twenty-four hours later, the cells were administered 10, 50, and 100?nmol/L insulin, and the cells on the glass coverslips were rinsed with PBS at 24?h and fixed with 95% ethanol for 30?min at room temperature. Next, the cells were DNA stained with 0.5?��g/mL DAPI (4?6?-diamidino-2-phenylindole dihydrochloride) (Sigma) solution for 15?min at room temperature in the dark. The coverslips were mounted and imaged with a Nikon Eclipse E800 fluorescent microscope. Apoptosis was quantified with Annexin V-FITC/PI apoptosis detection kit (KeyGen BioTECH) as previously described (Yao et?al. 2013). The data are reported as the mean?��?SEM. Analysis of variance (anova) was used to compare differences between the three different cell types and different concentrations of insulin. In all cases, differences were considered to be statistically significant when P?
