Fig. 8.

Accumulation of AZC in the proteomes of mouse and naked mole rat cells.A, the experimental design of proteomic experiments for measurement of AZC incorporation. Blue and red colors indicate ¹²C-proline and either AZC or ¹³C-proline, respectively. B, the number of AZC-containing peptides that were quantified in mouse cells treated with either DMSO or 2.5-mM AZC. C, incorporation of ¹³C-proline (left) and AZC (center, right) in mouse (red) and naked mole rat (blue) cells over time. The box plot representations are as described in Figure 1. The histogram to the right compares AZC incorporation in naked mole rats after 16 days of treatment with mouse cells after 8 days of treatment. D, comparisons of ¹³C-proline (left) and AZC (center, right) incorporation for individual peptides in mouse and naked mole rat cells. For pairwise comparisons (left, middle), peptides that could be quantified in both mouse and naked mole rat cells at 3 d were compared. Similar plots for other time points are shown in supplemental Fig. S8. For the violin plot (right), log₂ ratios of incorporation in mouse and naked mole rat cells are plotted for all peptides for which AZC incorporation could be measured in both species. E, the response to AZC and MG115 treatment in quiescent mouse (red) and naked mole rat (blue) cells. Cells were treated with 5-μM MG115 proteasome inhibitor and varying concentrations of AZC for 4 days. Cell viability represents the fraction of cells that excluded trypan blue, normalized to viability when treated solely with MG115 within each species. Error bars represent SD. ∗p-value < 0.05. F, accumulation of ubiquitinated proteins upon proteasomal inhibition and AZC treatment in mouse and naked mole rat cells. Cells were treated with 5-μM MG115 proteasome inhibitor and 2.5-mM AZC for 4 days, and the extracts were analyzed by Western blots using an anti-ubiquitin antibody. The right plot represents the quantification of the Western blot. The level of ubiquitin in MG115-treated samples for each species was normalized to the level of ubiquitin in the just AZC-treated mouse cells. AZC, azetidine-2-carboxylic acid.