Fig. 1. Result of mRNA-seq. Eight RNA samples including both conditions and gender (cold/control [cont] and male/female; n = 2 for each). A. Principal component analysis displaying the projection of the sample onto a two-dimensional scatter plot by the first and second principal components. B. Heatmap presenting DEGs for each sample (control and cold_2h). C. Scatter plot presenting the mean log₂ gene expression in the control and cold_2h groups. Green dots indicate excluded genes because of standard deviation > 1, blue dots indicate DEGs, and red dots indicate genes related to protein ubiquitination. D. Dot plots indicating the gene expression of trim63a, klhl38b, fbxo30a, and fbxo32.

Fig. 2. The result of qPCR. A–D. Ubiquitin-related genes trim63a, klhl38b, fbxo30a, and fbxo32 (n = 10–16). E–F. Protein anabolic-related genes myod1 and synpo2la (n = 10–16). All data are presented as relative gene expression in comparison to the reference gene gapdh. In the post-hoc test, ∗p < 0.05 and ∗∗p < 0.01 indicated significance in the comparisons between different time points, and †p < 0.05 and ††p < 0.01 indicated significance in the comparison between the cold and control [cont] groups at each time point.

Fig. 3. The result of western blotting A–C. Gel images of MuRF1 (trim63a), MAFbx (fbxo32), and gapdh. D and E. Time-course changes of MuRF1 and MAFbx (n = 6 for both conditions). All data are presented as relative gene expression in comparison to the reference protein gapdh. In the post-hoc test, ∗p < 0.05 indicated significance in the comparisons between different time points, and †p < 0.05 and ††p < 0.01 indicated significance in the comparisons between the cold and control [cont] groups at each time point.