Fig. 3: Alteration of proteostasis induced by glyoxal in MEF.

a Workflow for the analysis of proteome changes induced in MEF by GO. b Left, number of significant proteins (absolute log2 fold change>0.58 and q < 0.05) after 8 and 24 h GO treatment. Right, scatterplots comparing the log2 fold changes induced by GO at 8 or 24 h. Dashed lines represent the log2 fold change cut-offs used (±0.58). n = 5 (8 h), n = 4 (24 h). Pearson’s product-moment correlation, two-sided. c Distribution of protein fold changes for 2 mM GO (24 h) vs. control (Ctrl). Average values of n = 4 were compared, * p < 0.001, Wilcoxon rank sum test with continuity correction, two-sided. d Gene Set Enrichment Analysis (GSEA) for KEGG pathways based on protein fold changes induced by 2 mM GO (24 h). Normalized enrichment score (NES) indicates pathways enriched among proteins that increase (red) or decrease (blue) upon GO treatment (FDR < 0.05). All quantified proteins were ranked according to their log2 fold change and used as input for GSEA. e Left, boxplot of fold changes in expression of members of the 26S proteasome after GO treatment (24 h). * p < 0.01, Wilcoxon Rank Sum test with continuity correction, two-sided. Right, proteasome chymotrypsin-like (CT-L) activity in MEF treated with GO (24 h). n = 4, mean + SD, * p < 0.05, one-way ANOVA using Geisser-Greenhouse correction. f Immunoblot for mono- and polyubiquitinated proteins from MEF treated with GO (24 h). Positive control: proteasome inhibitors MG132 (20 µM, 6 h) or epoxomicin (Epox, 10 nM, 96 h). n = 4, mean ± SD, * p < 0.05, one-way ANOVA using Geisser-Greenhouse correction. g Heatmap representing the melting point temperature of members of 26S proteasome using thermal proteome profiling. h Left, proteasome activity assay using native gel electrophoresis (top) after incubation of purified proteasome with GO (1 mM, 0, 1, 2, and 4 h, 37 °C). Immunoblot for CML (middle) and proteasome subunits α1-7 (bottom). Middle, quantification of CML-modification. Right, proteasome chymotrypsin-like activity (CT-L) normalized to α1-7 abundance. Positive control: purified proteasome incubated with MG132 (100 µM, 4 h, 37 °C). n = 4, mean + SD, * p < 0.05, one-way ANOVA. Source data are provided as a Source Data file. Specific p values are listed in Supplementary Data 6. Related to Supplementary Fig. 3, 4 and Supplementary Data 3.