gcb12592-sup-0001-SupportingInformation.docxWord document2783K

Data S1. Materials and methods.

Figure S1. Graphical illustration of the experimental design.

Figure S2. Principle component analysis (PCA). All the samples and replicates are plotted in a three-dimensional space using three principal components (PC) capturing 65.3% of total variability. This image was constructed using Partek Genomics SuiteTM software.

Figure S3. Venn diagram of all differentially expressed genes. The Venn diagram presents differentially expressed genes in the 28 (t1 vs. c1), 32 (t2vs. c2) and 34 °C (t3 vs. c3) trials. The number of genes is indicated within each field.

Figure S4. The relative expression of selected genes assessed via quantitative real-time PCR (ddCt). The expression levels of (A) dnajc3 (DnaJ homolog subfamily C member 3), (B) pdia6 (protein disulfide-isomerase A6) (C) Collagen alpha and (D) Carbonic anhydrase 6, in treatments 1 (28 °C) and 3 (34 °C) (indicted in black) and their corresponding controls (indicted in gray) are shown. Each dCt was normalized to a housekeeping gene: 60S Ribosomal protein L22. Error bars indicate the standard error.

Figure S5. Fold change values of selected up-regulated (A) and down-regulated (B) genes. The fold changes of genes for ER stress and protein folding in the ER and proteasomal ubiquitin-mediated proteolysis (A), and genes for extracellular matrix organization, Wnt signaling, Notch signaling and actin cytoskeleton organization (B) are shown with respect to temperature treatments 1, 2 and 3. The color-scale legend indicating the relative fold change is shown on the right. This figure was constructed using expander software.

Figure S6. Venn diagram of total differentially expressed genes in preliminary 32 °C and current 32 and 34 °C trials. The number of genes is indicated within each field.

Table S1. A list of 163 differentially expressed genes – ‘Common genes’ – either up- or down-regulated, at all three sampled temperatures. −1 and 1 indicate significant (FDR P < 0.05) down- and up-regulation, respectively.

Table S2. Down-regulated genes from the skeletal organic matrix (SOM) predicted proteins identified by Drake et al. 2013 proteomic analysis, in the 28°C (t1 vs. c1), 32°C (t2vs. c2) and 34°C (t3 vs. c3) trials. [DOWNWARDS ARROW] and [UPWARDS ARROW] indicate significant (FDR p<0.05) down-regulated and up-regulated genes, respectively.

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