The identified protein groups were compared using Venn diagrams (http://bioinformatics.psb.ugent.be/webtools/Venn/ (accessed: December 2020)). Gene ontology (GO) analysis was performed using the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 [33 (link)], with the whole Homo sapiens genome as statistical background. GO analysis was performed using a Fisher’s exact test followed by FDR multiple testing correction. The identified protein groups were compared with the EV markers present in the ExoCarta Top 100 database [55 ,56 (link)], with the Human Brain Proteome [57 ] and with the Parkinson Disease KEGG Pathway [43 ]. These comparisons were performed by string search of the protein names. Protein datasets were compared using the Pearson correlation coefficient to assess reproducibility. All proteomics data were analyzed using Perseus 1.5 software [41 (link)], Microsoft Excel, GraphPad Prism v5 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com ). For quantitative analysis, common contaminants were removed prior to data normalization, and the raw protein intensities were then log2 transformed and median normalized.
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17 protocols using graph pad prism v 5 for windows
1
Comparative Proteomics Analysis of Extracellular Vesicles
2
Quantitative PCR Analysis Protocol
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The values represent the average of at least three independent experiments, significance being (ns,*,**,***,&) non-significant, P < 0.01, 0.001, 0.0001, 0.0005. The RT-qPCR data were analyzed using the 2-ΔΔCT method. GraphPad Prism v.5 for Windows (GraphPad Software, San Diego, CA, USA) was used to analyze results, using Student’s t-test and Kruskal–Wallis test.
3
Statistical Analysis of Neurotransmission
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Data are presented as means ± SEM of n experiments. Statistical significance of differences between PI fluorescence intensities or Western blot optical densities was evaluated by performing one-way ANOVA followed by Dunnet’s test for multiple comparisons or by the Kolmogorov–Smirnov test (sEPSC recordings). All statistical calculations were performed using GRAPH-PAD PRISM v. 5 for Windows (GraphPad Software, San Diego, CA, United States). A probability value (P) of < 0.05 was considered significant.
4
Evaluating Cell Cytotoxicity Profiles
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Data are presented as means ± SEM of n experiments. The statistical significance of differences between PI fluorescence intensities, LDH release, or Western blot optical densities was analyzed using one-way ANOVA with a post hoc Tukey’s w-test for multiple comparisons. All statistical calculations were performed using GRAPH-PAD PRISM v. 5 for Windows (GraphPad Software, San Diego, CA, USA). A probability value p < 0.05 was considered significant.
5
Quantifying Cell Fluorescence Intensities
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Data are presented as means ± SEM of n experiments from independent cell preparations. Statistical significance of differences between PI fluorescence intensities, immunostaining, or Western blot optical densities was evaluated by performing one-way ANOVA followed by Tukey’s w test for multiple comparisons. All statistical calculations were performed using GRAPH-PAD PRISM v. 5 for Windows (GraphPad Software, San Diego, CA, USA). A probability value (P) of < 0.05 was considered significant.
6
Multivariate Analysis of Experimental Data
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All data were expressed as means ± standard deviation unless otherwise specified. Statistical analysis was performed using GraphPad Prism v.5 for Windows (GraphPad Software Inc., San Diego, CA, USA). Intergroup differences for continuous variables were assessed by multivariate ANOVA. P<0.05 was considered statistically significant.
7
Multivariate Analysis of Environmental Pollutants
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Statistical analyses were performed using GraphPad Prism v5 for Windows (GraphPad Software, La Jolla, CA, USA). One-way ANOVA with a Tukey multi comparison test was used to establish statistical differences between the groups. The mean of each data set was compared between them using a p < 0.05. The Principal Component Analysis (PCA) was performed considering all parameters determined in the study (PAHs, metals, and biological activity) and using the “The Unscrambler” software (version 9.7, CAMO PROCESS AS, Oslo, Norway) as described before [58 (link)].
Algorithmic prediction for the determination of PAHs was performed using MATLAB (MathWorks Inc 2008) program with the algorithms “unfolded partial least squares with residual bilinearization” (U-PLS/RBL) and “parallel factor analysis” (PARAFAC). Both algorithms were implemented using the graphical interface MVC2. Routines to perform analyses are available on the internethttp://www.models.kvl.dk/algorithms as is the graphical interface used MVC2 (http://www.chemometry.com ).
Algorithmic prediction for the determination of PAHs was performed using MATLAB (MathWorks Inc 2008) program with the algorithms “unfolded partial least squares with residual bilinearization” (U-PLS/RBL) and “parallel factor analysis” (PARAFAC). Both algorithms were implemented using the graphical interface MVC2. Routines to perform analyses are available on the internet
8
Treadmill Run Substrate Oxidation Analysis
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Substrate oxidation during the 1 h treadmill run was calculated with the proposed equations from Jeukendrup and Wallis [25 (link)] for exercise with moderate intensity (50% O2max) to high intensity (75% O2max). For analysis of the physiological (i.e., heart rate, minute ventilation, oxygen uptake, carbon dioxide production) and metabolic parameters (i.e., fat oxidation, carbohydrate oxidation, and respiratory exchange ratio) in the control and New Zealand blackcurrant extract conditions, a paired two-tailed t-test was used (GraphPad Prism v5 for Windows, GraphPad software, San Diego, CA, USA) for the 15 min (i.e., 4 time points) during the 1 h treadmill run. Core temperature recordings were averaged for 15 min time periods and analyzed with a paired two-tailed t-test. Data are reported as mean ± SD, calculated from the 15 min (i.e., 4 time points) measurements during the 1 h treadmill run. Significance was accepted at p < 0.05.
9
Age and Sex Differences in Neurobehavioral Measures
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Data were analyzed using GraphPad Prism v.5 for Windows (GraphPad Software, San Diego, CA), and are presented as the mean ± SEM. Differences between the control, NH, and H groups, and age (1, 5, and 11 months) were analyzed using a two‐way analysis of variance (ANOVA) with the factors group (PG), age (PA), and their interaction (PG × A). Differences between males and females, and groups, were assessed at each time point using two‐way ANOVA, with the factors group (PG), sex (PS), and their interaction (PG × S). Body weights were analyzed using two‐way ANOVA with repeated measures. To determine differences between individual groups, each two‐way ANOVA was followed by a Bonferroni post hoc test. Statistical significance was accepted at the level of P < 0.05.
10
Cell Viability and Uptake Analysis
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The experiments were repeated three times and results expressed as a mean ± standard deviation. Statistical significance of hCMEC/D cell viability and cellular uptake was analyzed using one-way ANOVA followed by the post hoc Tukey’s w-test for multiple comparisons. All statistical calculations were performed using GRAPH-PAD PRISM v. 5 for Windows (GraphPad Software, San Diego, CA, USA). A probability value (p) of <0.05 was considered significant.
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