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. 2023 Aug 7;12:e88875. doi: 10.7554/eLife.88875

Figure 2. Loss of Hsp31 paralogs aggravates the glycation of DNA and RNA, affecting translational activity.

(A, B) Immunodetection of DNA using dot-blot assay. Three µg of total genomic DNA from respective strains treated overnight with 15 mM GO was dotted on nitrocellulose membrane and probed with anti-CML antibody. (C, D) RNA glycation profile. Global RNA extracted from cells incubated with 15 mM GO were dotted and analyzed using anti-CML antibody. (E) Polysome profiling. Untreated (-GO) and treated (+GO) WT and ΔQ cells with GO were subjected to polysome profiling. (F) Ratio of polysomes to monosomes (P/M). The area occupied by the polysome and the monosome peak was determined using Origin 8.0 for the respective samples, and the ratios were plotted. WT (+GO) was compared with WT (-GO), and ΔQ (-GO) was compared with ΔQ (+GO). (G–J) Estimation of MAGE-modified nucleic acids. Strains lacking Hsp31 paralogs were treated with 10 mM MG for 12 hr, and the genomic DNA and RNA were dotted on the membrane and probed with anti-MAGE antibody. The relative intensity of dots representing the glycation levels was calculated and compared to WT. One-way ANOVA with Dunnet’s multiple comparisons test was used to determine significance from three independent biological replicates, *, p≤0.05; **, p≤0.01; ***, p≤0.001; NS, not significant.

Figure 2—source data 1. Source data for Figure 2A contains raw image of western blot and densitometric values for graph.
Figure 2—source data 2. Source data for Figure 2B contains raw image of western blot and densitometric values for graph.
Figure 2—source data 3. Source data for Figure 2C contains raw image of western blot and densitometric values for graph.
Figure 2—source data 4. Source data for Figure 2D contains raw image of western blot and densitometric values for graph.
Figure 2—source data 5. Source data for Figure 2E contains densitometric values for graph.
Figure 2—source data 6. Source data for Figure 2F contains densitometric values for graph.
Figure 2—source data 7. Source data for Figure 2G contains raw image of western blot and densitometric values for graph.
Figure 2—source data 8. Source data for Figure 2H contains raw image of western blot and densitometric values for graph.
Figure 2—source data 9. Source data for Figure 2I contains raw image of western blot and densitometric values for graph.
Figure 2—source data 10. Source data for Figure 2J contains raw image of western blot and densitometric values for graph.

Figure 2.

Figure 2—figure supplement 1. Glycation profile of DNA in the absence of GO stress.

Figure 2—figure supplement 1.

(A, B) DNA extracted from stationary phase cells was dotted (3 µg) on nitrocellulose membrane and probed with anti-CML to determine the glycation status.
Figure 2—figure supplement 1—source data 1. Source data for Figure 2—figure supplement 1A contains raw image of western blot.
Figure 2—figure supplement 1—source data 2. Source data for Figure 2—figure supplement 1B contains raw image of western blot.