206 Chapter 8 was performed using the EditObjectsManually module to accurately bin cardiomyocyte and non-myocyte nuclei. To identify the nuclear PPARA signal of the cardiomyocyte nuclei, a mask was first made on the PPARA channel using the MaskImage module resulting in an image showing only the PPARA signal within the cardiomyocyte nuclei. The signal was then identified using the IdentifyPrimaryObjects module. The MaskObjects module was then used to assign each PPARA signal to a single parent nucleus. Intensity measurements were made for both the cardiomyocyte nuclei and the PPARA signal using the MeasureObjectIntensity module. Data for the PPARA signal per individual nuclei was then exported to a CSV file using the ExporttoSpreadsheet module. Full details of the pipeline can be seen in the attached CellProfiler file. All data was represented as Mean ± SD and two-way ANOVA was used for statistical analysis (*p < 0.05; **p < 0.01; ***p < 0.001, 0.001 and ****P ≤.0001). The list of antibodies is shown in Table S8. Differentiation of hiPSC into cardiomyocytes. Reprogrammed Sendai (kit) accordingly as described before.15 Briefly, hiPSCs (>p20 <p50) were grown to ~90% confluence in 6 wells format and maintained in E8 medium for at least three passages before starting cardiac lineage differentiation. Upon differentiation, the medium was changed to medium without insulin (INS- medium), as insulin inhibits cardiac mesoderm formation. On day zero (day 0), 3 ml/well INS- medium was supplemented with 6-8 μM CHIR99021 (Selleck Chemicals). After one night of incubation, an additional 2 ml of the INS- medium was carefully added (day 1), and an additional 1 ml of the INS- medium was added on day 2. On day 3, the medium was replaced by a 3 ml/well INS- medium supplemented with 2 μM Wnt-C59 (Tocris Bioscience). Afterwards, the culturing medium was changed every other day and contracting cells were generally seen between day 7 and day 9. After day 9, the INS- medium was changed to 3 ml/well INS+ medium. To metabolically select and purify hiPSC-CMs, INS+ was replaced with a purification medium (without D-glucose) for 2 days. Purified hiPSC-CMs were continuously cultured in INS+ for 2 days to recover. On day 15 hiPSC-CMs were detached by 10 mins incubation of 10X TrypLE select 10X (Thermo Fisher) at 37°C. Detached cells in TrypLE solution were flushed using INS+ medium and cell pellets were obtained by centrifuging at 300g for 3 minutes. After aspiration, hiPSC-CMs were resuspended in INS+ medium supplemented with 5 uM ROCK inhibitor and 10% KO-serum (Thermo Fisher) and filtered through a 100 µm pore filter (Corning). Approximately three million hiPSC-CMs were re-plated into Matrigel-coated wells in a 6-well plate and further cultured for 2 days in INS+ medium for recovery. To ensure the purity of hiPSC-CMs, they were cultured in a purification medium for 2 days before all cell-based experiments in this study (e.g. RNA-seq and Seahorse assays). Media used for the cultivation of iPSC-CMs. To mature hiPSC-CMs, we used the maturation medium developed in the Mercola lab.16 After the 2nd purification as indicated above,
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