Prism v6.0d

To analyze DTEHV fatigue behavior over time, a linear regression analysis was performed (Prism V6.0d, Graphpad, USA) on the closing volume, leakage volume, cardiac output, and the effective orifice area, with a slope being significantly non-zero for p < 0.05. Time points were averaged, and represented by their mean ± standard deviation.
For the biomechanical analysis, samples were averaged per valve, and represented by their mean ± standard deviation. The presence of anisotropy was defined as a significant difference (p < 0.05) between the obtained stiffnesses in both radial and circumferential direction, analyzed using a paired student t test (Prism V6.0d, Graphpad, USA).

Image sequences were analyzed (registration, background subtraction, ROI intensity versus time analysis) using open-source FIJI software (http://fiji.sc/Fiji). The numerical time series data were analyzed using the IgorPro package (Wavemetrics). Statistical significance of the differences between paired or unpaired samples was tested using Friedman or Kruskall-Wallis tests, respectively, with Nemenyi post hoc analysis, as implemented in the R package (R Development Core Team, 2016 ). Basal pH_i was calculated by taking the mean of the first 15 SNARF-5F ratio values from each cell. After glyceraldehyde and 20 mM glucose application, pH_i was calculated by taking a mean of the values when the SNARF-5F ratio reached a nadir. Differences with p < 0.05 were considered significant. Cells that were not active at 3 mM glucose and that responded to high glucose with the characteristic [Ca²⁺]_i oscillations were taken as β cells.
All data are given as mean ± SEM unless indicated that they are mean ± SD. Other than for image analysis, as indicated earlier, statistical significance was determined with significance set at <0.05, using either ANOVA with Tukey’s multiple comparison or Student’s t test (where indicated). Statistical significance was determined using GraphPad Prism v.6.0d (GraphPad Software, La Jolla, CA, USA; http://www.graphpad.com).

Pipelines for primary analysis (filtering and alignment to the reference genome of the raw reads) and secondary analysis (expression quantification, differential gene expression and peak calling) have been integrated in the HTS-flow system^{89 (link)}. Bioinformatic and statistical analysis were performed using R with Bioconductor packages and comEpiTools packages^{90 ,91} . Motif analysis was performed using CentriMo software^{92 (link)}. Intron analysis was performed with the INSPEcT tool^{93 (link),94 (link)}. Gene lists from RNA and ChIP sequencing were analysed in Enrichr⁹⁵ . Statistical analyses for IHC and qRT-PCR were performed using GraphPad Prism v6.0d (GraphPad Software, Inc., San Diego, CA, USA) as indicated with P ≤ 0.05 considered to be statistically significant. Venn diagrams were draw in the interactive tool Venny [https://bioinfogp.cnb.csic.es/tools/venny/index.html].