Prism 7.0b

Statistical analyses were done by GraphPad Prism 7.0b software.

Graphs were generated, and data were analyzed using GraphPad Prism 7.0b software (MacKiev). Statistical analysis was performed using a Student’s t test. A P value of <0.05 was considered significant.

Glycosaminoglycan-binding ELISA plates (BD Life Sciences, Swindon, UK) were coated with suramin or its analogs (10 µg/ml in Tris-buffered saline, 18 hours, 25°C) (Mahoney et al., 2004 (link)) and wells were blocked with 0.2% gelatin in PBS (1 hour, 37°C). Wells were washed in PBS containing 0.1% Tween 20 after this and every subsequent step. Purified FLAG-tagged human TIMP-3 (0.4–50 nM) in blocking solution was applied to wells (3 hours, 37°C), and binding was detected with anti-FLAG M2 primary antibody and anti-mouse horseradish peroxidase–conjugated secondary antibody. 3,3′,5,5′-Tetramethylbenzidine (Becton Dickinson, Swindon, UK) substrate was added, the reaction was stopped when appropriate by adding 2 N H₂SO₄, and absorbance at 450 nm was measured using a FLUOstar Omega microplate reader (BMG Labtech, Aylesbury, Buckinghamshire, UK). Data (mean ± S.E., n = 3 technical repeats) were analyzed using Prism 7.0b software (GraphPad Software, La Jolla, CA) and EC₅₀ values determined using a one-site specific binding model. Flat planar dimensions of suramin analogs were estimated using ICM-Pro software (Molsoft LLC, San Diego, CA).

Statistical analyses were done by GraphPad Prism 7.0b software. Experiments were repeated independently on multiple flies as mentioned in the text and figure legends.

Patient demographics were compared by the Mann–Whitney or Fisher’s exact test as appropriate. Differences in the abundance of fecal metabolites between control cats and cats with CE were evaluated using a Mann–Whitney test. A post hoc analysis to assess differences in the abundance of metabolites between the subgroups (i.e., controls, IBD, SCL) was performed by Dunn’s test. Statistical significance was set at p < 0.05. Results were adjusted by False Discovery Rate (FDR) and reported as the q-value where appropriate. Univariate analysis was performed using Prism 7.0b (Graph Pad Software, La Jolla, CA) and JMP Pro 14.1.0 (SAS Institute Inc., Cary, NC).
Multivariate analysis was performed using MetaboAnalyst^{66 (link)}. Data was mean centered and divided by the standard deviation of each variable. PCA and hierarchical clustering was performed and a heatmap was created as a visual aid for the dendrogram. Random forest regression analysis was used to evaluate the classification performance of metabolomics.