Fig 1: Donor age affects the abundance of selected proteins identified in equine mesenchymal stromal cell (MSC) conditioned media (CM). (A) Cluster members identified with fuzzy time course clustering. Graphs show clusters of proteins with similar abundance patterns and change in their abundance value with donor age. (B) Scatterplots showing change in CM protein abundance, measured by quantitative mass spectrometry, with MSC donor age. Dots represent biological replicates, colors tissue type used for MSC isolation, lines the mean protein abundance predicted by generalized linear model (GLM) and shades 95% CI. Proteins with the strongest relationship between age and abundance (P-value <.05, q-value <.2) are shown: collagen triple helix repeat-containing protein 1 (CTHRC1), lysyl oxidase (LOX), transforming growth factor beta induced (TGFBI), cadherin 11 (CDH11). (C) Western blot analysis of the abundance of CTHRC1 in CM from bone marrow-derived (BMSC) and adipose-derived (ASC) equine MSCs, isolated from donors of different age (n = 6). Heatmap bar shows the abundance of CTHRC1 (LFQ intensity) measured in CM by mass spectrometry. Fibronectin (FBN) was included as loading control (i). Density of CTHRC1 protein bands relative to FBN were measured with ImageJ and plotted against LFQ intensity of CTHRC1 in CM from the same MSC samples (ii). (D) Scatterplot showing enzymatic activity of LOX in MSC CM (n = 27), measured with a commercial fluorescence assay. The y-axis shows the difference in fluorescence intensity (RFU, relative fluorescence units), normalized to total protein concentration, between 30 minutes and 10 minutes incubation time (delta RFU). (E) Scatterplot showing change in MCP-1 concentration in MSC CM in relation to donor age. Dots represent biological replicates, lines the mean protein abundance predicted by GLM, and shades 95% CI.