Data shown are the means ±SEM. Data between 2 groups were analyzed by unpaired t test (Prism 5.0; graph Pad Software, San Diego, CA, USA) if the data were in Gaussian distribution and had equal variance, or by unpaired t test with Welch’s correction (Prism 5.0; graph Pad Software) if the data were in Gaussian distribution but with unequal variance, or by nonparametric test (Mann-Whitney U test, Prism 6.0; graph Pad Software) if the data were not normally distributed. Data among more than 2 groups were analyzed by the one-way ANOVA followed by Dennett multiple comparisons (Prism 5.0; graph Pad Software) if the data were in Gaussian distribution and had equal variance or analyzed by nonparametric Kruskal-Wallis one-way analysis with Dunn multiple comparison post hoc test (Prism 5.0; graph Pad Software) if the data were not normally distributed. The Gaussian distribution of data was analyzed by D’Agostino‐Pearson omnibus normality test (Prism 5.0; graph Pad Software) and Kolmogorov‐Smirnov test (Prism 5.0; graph Pad Software). The variance of data was analyzed by homogeneity of variance test (SPSS 22.0) or Brown‐Forsythe test (Prism 6.0; graph Pad Software). Statistical details of all experiments can be found in the figure legends and significance is described in the figure legends as: * p < 0.05, ** p < 0.01.
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Lab products found in correlation
Spss 22, by IBM (145 mentions)
Spss 20, by IBM (140 mentions)
Sas 9, by SAS Institute (130 mentions)
Spss 19, by IBM (130 mentions)
Spss 21, by IBM (88 mentions)
Spss version 20, by IBM (66 mentions)
Sas version 9, by SAS Institute (64 mentions)
Spss statistics 20, by IBM (58 mentions)
Spss statistic, by IBM (50 mentions)
Spss version 19, by IBM (48 mentions)
Spss version 22, by IBM (47 mentions)
Clampfit 10, by Molecular Devices (46 mentions)
Microplate reader, by Thermo Fisher Scientific (43 mentions)
Spss version 17, by IBM (43 mentions)
Sigmaplot 12, by Grafiti LLC (42 mentions)
Spss version 21, by IBM (42 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (40 mentions)
Matlab, by MathWorks (39 mentions)
Spss version 18, by IBM (39 mentions)
Spss statistics 22, by IBM (37 mentions)
39 065 protocols using prism 5
1
Statistical Analysis of Experimental Data
2
Statistical Analysis of Cell-based Experiments
3
Comparing Survival and Melanization Curves
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To compare survival curves, the Log-rank test was performed with GraphPad Prism 5 (version 5.03, GraphPad Inc.) To determine if there was a statistical difference across the different groups in terms of melanization and grain counts, the Kruskal-Wallis test was performed with GraphPad Prism 5. If a difference was found with the Kruskal-Wallis test, pair-wise comparisons were made between the PBS treated groups and the different antifungal treated groups with the Mann-Whitney U test with GraphPad Prism 5 to determine differences in melanization or CFU count. A p-value smaller than 0.05 was deemed significant. All negative values after normalization were refigured to zero for statistical analysis. To determine the statistical difference in the total number and sizes of grains between the treated and non-treated groups, a Mann-Whitney test was performed with GraphPad Prism 5. A p-value smaller than 0.05 was deemed significant.
4
Radioligand Binding Assay Analysis
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Data generated from binding assays were analyzed using Prism 5.02 (GraphPad Software, Inc., San Diego, CA, USA). Data points from radioligand inhibition binding curve were fitted to models using nonlinear regression equation to determine inhibitor potency (IC50) estimates, which were then converted to Ki values [35 (link)] as appropriate.
Data from one-point kinetic experiments were analyzed in order to obtain estimates of the affinity of an allosteric ligand for the [3H]-NMS occupied receptor (log Kocc) in a single step, using an equation introduced in GraphPad Prism 5.02 [33 (link),34 (link)].
Radioligand dissociation rates in absence or in presence of the allosteric modulator were analyzed by non linear regression according to the equation for mono-exponential decay using GraphPad Prism 5.02.
All the results are expressed as means ± SEM obtained from 3-4 independent experiments each one performed in duplicate.
Data from one-point kinetic experiments were analyzed in order to obtain estimates of the affinity of an allosteric ligand for the [3H]-NMS occupied receptor (log Kocc) in a single step, using an equation introduced in GraphPad Prism 5.02 [33 (link),34 (link)].
Radioligand dissociation rates in absence or in presence of the allosteric modulator were analyzed by non linear regression according to the equation for mono-exponential decay using GraphPad Prism 5.02.
All the results are expressed as means ± SEM obtained from 3-4 independent experiments each one performed in duplicate.
5
BACE-1 Inhibitory Assay in Drosophila
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Concentrations of the test compounds causing 50% loss of BACE-1 (IC50) were calculated using GraphPad Prism 5.1 software (San Diego, CA). The IC50 values for each D. melanogaster line and their treatments were considered to be significantly different from one another when their 95% confidence limits (CL) failed to overlap. All data are presented as mean ± standard error, and the significance between means was determined using one-way or two-way analysis of variance (ANOVA) statistical test (GraphPad Prism 5.1 software; San Diego, CA). Statistical analysis for survival data were carried out using the Bonferroni post tests (GraphPad Prism 5.1 software).
6
Kinetic and Inhibition Enzyme Assays
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Enzyme kinetic parameters were determined using GraphPad Prism 5.00 for Windows, GraphPad Software (San Diego, USA; RRID:SCR_002798) and fitted to the Michaelis–Menten equation. IC50 was calculated using the log (inhibitor) versus response function of GraphPad Prism 5.00 for Windows, GraphPad Software (San Diego, USA). The Pearson correlation coefficient was calculated for the specific probe substrates and flucloxacillin 5′‐hydroxylation using GraphPad Prism 5.00 for Windows, GraphPad Software (San Diego, USA). A two‐tailed P‐value was used with a confidence interval of 95%. The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018 ). However, randomization of samples was deemed unnecessary since no systematic bias was observed related to the sample position or in the order of sample analysis by LC–MS. Blinding was not undertaken since prior knowledge of sample content was not expected to introduce bias of the measurements. Also, data acquisition and data analysis were performed by different individuals.
7
Embryonic Development Evaluation Protocol
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Sample size was determined as indicated in the figures and specific statistical method was not used. Dead cells and embryos were excluded from all experimental analysis. Embryos which have mis-targeted injection also were excluded (the target injection was confirmed by co-injection of lineage tracer (GFP RNAs, GFD or RFD). All experiments were performed blinded with order of testing randomised. ImageJ program was used for all quantification. All experiments were performed for at least three independent times. Normality of data was tested using Kolmogorov–Smirnov’s test, D’Agostino and Pearson omnibus normality test and Shapiro–Wilk normality test using Prism5. The data were considered normal if found as normal by all three tests. Data sets following a normal distribution were compared with Student’s t test (two-tailed, unequal variances) or a one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons post-test in Prism5. The data that did not follow a normal distribution were compared using Mann–Whitney’s test or a non-parametric ANOVA (Kruskal–Wallis with Dunn’s multiple comparisons post-test) using Prism5. Cross-comparisons were performed only if the overall P value of the ANOVA was <0.05.; error bars: s.d.
8
Kinetic Analysis of MIPS and MIPP Enzymes
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The effect of substrate concentration and kinetic analyses for partially purified MIPS were carried out using D-glucose-6phosphate (substrate) concentrations in the range of 0.0 to 10.0 mM at an interval of 1.0 mM. All data regarding specific activity of MIPS corresponding to the respective substrate concentration were analyzed by means of non linear regression kinetics using Prism 5 (Grapg pad) software package. Similar experiments for MIPP were also carried out using D-myo-inositol-1-phosphate (substrate) concentrations in the range of 0.0 to 1.0 mM at an interval of 0.1 mM. The data regarding specific activities of MIPP corresponding with respective substrate concentrations were also analyzed using Prism 5. The effect of coenzyme concentration (NAD) and kinetic analysis for MIPS was carried out using NAD in the range of 0.0 to 1.0 mM (maintaining an interval of 0.1 mM). Enzyme incubation and assay was carried out following methods described earlier. Specific activities of MIPS corresponding with the respective coenzyme concentrations were also analyzed using Prism 5 (Grapg pad). All results were obtained directly following software analyses.
9
Statistical Analysis of Quantitative PCR Data
10
Yeast Cell Size, Growth, and Budding
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For cell size measurement, yeast strains were cultured in the YPD liquid medium at 28 °C or 37 °C, with shaking speed of 180 rpm for 48 h. Cells were harvested and the cell size was measured with a Nikon Eclipse 80i fluorescence microscope (Nikon, Tokyo, Japan). Forty cells were removed randomly from each sample to measure cell size. Data analysis was performed using GraphPad Prism 5 software. Unpaired t-test was used for statistical analysis.
For growth curves, overnight cultures were transferred to fresh YPD liquid medium and continued to culture at 28 °C or 37 °C for 48 h. Absorbance was measured at 600 nm every 2 h, and the initial absorbance of OD600 was 0.2. Data were processed using GraphPad Prism 5 software.
For calculation of the budding rate, overnight cultures were transferred to the fresh YPD medium and continued to culture at 28 °C or 37 °C for 16 h (mid-log phase). The total number of yeast cells and the number of budding cells were counted under a microscope. The assay was performed three times. Budding rate was performed using GraphPad Prism 5 software. Unpaired t-test was used for statistical analysis.
For growth curves, overnight cultures were transferred to fresh YPD liquid medium and continued to culture at 28 °C or 37 °C for 48 h. Absorbance was measured at 600 nm every 2 h, and the initial absorbance of OD600 was 0.2. Data were processed using GraphPad Prism 5 software.
For calculation of the budding rate, overnight cultures were transferred to the fresh YPD medium and continued to culture at 28 °C or 37 °C for 16 h (mid-log phase). The total number of yeast cells and the number of budding cells were counted under a microscope. The assay was performed three times. Budding rate was performed using GraphPad Prism 5 software. Unpaired t-test was used for statistical analysis.
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