Fig 2: Metabolic imaging and glucose tracing in GS-deficient liver.a, Serial sections of frozen liver samples from wt/wt and ?/? female mice. Left, IHC staining for GS and OAT. Right, MS metabolic imaging of glutamine and glutamate. Data were normalized with the root mean square method; scale bar, 1 mm. Images are representative of n = 3 livers per genotype (Extended Data Fig. 1d,e). b, Quantification of glutamine and glutamate in the regions of interest shown in a (n = 1 wt/wt, n = 1 ?/?). Boxes have bounds at the 25th to 75th percentiles, the lines represent the medians and whiskers show the 5th to 95th percentiles; each data point represents the relative intensity of one pixel. Data were analzyed by two-tailed Student’s t-test. c–f, Levels of citrate (c), a-ketoglutarate (aKG; d), succinate (e) and malate (f) in the livers of wt/wt (n = 9) and ?/? (n = 12) mice. g, Relative levels of 13C2 isotopolog in the livers of wt/wt (n = 4) and ?/? (n = 5) mice administered U-13C6-glucose. Data were analyzed by two-tailed Student’s t-test. Bars represent mean ± s.e.m., and each circle represents data from a single mouse.Source data

Fig 3: Validation of candidate ClpXP substrates identified by TAILS.A, Putative ClpXP substrates with increased N termini abundance. MTS (yellow) and mature proteins (blue) are shown with the starting positions of the accumulating N termini indicated by white numbers. Above the proteins, cleavage windows are stated with the difference in abundance (log2(Clpp−/−/wt)). Amino acids at the P1 position preceding the cleavage site are highlighted in red, the detected peptide sequence is underlined. B, Western blots of steady state protein levels in heart lysates. HSC70 and CALNEXIN were used as loading controls for the respective blots. C, Relative gene expression with qPCR of Oat, Lonp1 and Hspa9. D, CHX chase experiment of HSPA9 in MEFs. HSC70 was used as loading control. E, CHX chase experiment of OAT in MEFs. HSC70 was used as loading control.

Fig 4: Patterns of liver transduction Liver section from wild-type C57BL/6N mice systemically injected with an AAV8 vector driving GFP expression under the control of the TBG promoter (AAV-TBG-GFP) or under the control of the OAT regulatory sequence (AAV-OATrs-GFP). Representative images from experimental groups including at least n = 4 mice per group are shown. Scale bar: 20 µm.Western Blot analysis of lysates from livers of wild-type C57BL/6N mice injected with AAV-TBG-GFP (n = 4) or AAV-OATrs-GFP (n = 5) vectors.Representative immunofluorescence images of livers from wild-type C57BL/6N mice (n = 4) injected with AAV-TBG-GFP. OAT staining is show in red and GFP in green, colocalization of the signal is shown in yellow. Scale bar: 20 µm.

Fig 5: OAT gene transfer in Oat rhg pigmented mice injected at 6 weeks of age improves retinal phenotype Plasma ornithine concentrations in Oat rhg mice injected with AAV-OAT (red circles, n = 5) or AAV-GFP (black circles, n = 3) vectors. Averages ± SEM are shown; the two-way ANOVA test was used to perform a statistical comparison between groups; **P < 0.01; ***P < 0.001, ****P < 0.0001.Body weights of Oat rhg mice injected at 6 weeks of age; the arrow indicates the time of the injections with AAV-OAT (red circles, n = 5) or AAV-GFP (black circles, n = 3) vectors; data are shown as means ± SEM; Two-way ANOVA test was used for groups comparison.a- and b-waves amplitudes recorded in scotopic conditions plotted as a function of light intensity (log cd * s/m2) in eyes of 11-month-old Oat rhg mice injected with AAV-OAT (n = 9, red circles) or AAV-GFP (n = 4, black circles) vectors. Wild-type (WT) mice were used as controls (n = 10, blue circles). All data are shown as averages ± SEM. Two-way ANOVA test was used for comparison between groups. **P < 0.01; ***P < 0.001, ****P < 0.0001.Plasma ornithine and lysine concentrations of Oat rhg mice at 12 months post-injection measured by HPLC. One-way ANOVA test with Tukey correction was used for comparison between groups; data are shown as averages ± SEM. *P < 0.05; **P < 0.01; ****P < 0.0001.Ornithine concentrations in the eyes of mice injected with AAV-OAT compared with eyes of either wild-type or AAV-GFP controls. Data are shown as z-scores. Means ± SEM are shown. One-way ANOVA with False Discovery Rate correction was used for comparison between groups. **P < 0.01, ****P < 0.0001.Western blotting for OAT and GFP on livers of Oat rhg mice harvested 12 months after the injections of AAV-OAT or AAV-GFP vectors. A WT mouse was included as control, and Vinculin was used as loading control.Morphological analysis of Oat rhg mice retinas 12 months post-injection of AAV-GFP or AAV-OAT. An age-matched WT mouse is shown as control (left panel). Semi-thin sections (40×). Abbreviations: ns, not statistically significant difference; RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; WT, wild-type.