Fig 2: SIRT5 promotes CRC growth by activating TKT to sustain the nucleotide pool in vivo.a, b A surgical orthotopic mouse model by injecting luciferase-transfected HCT116 cells stably expressing the non-target control (NTC) shRNA or SIRT5 shRNAs into the cecum of nude mice was generated. A bioluminescence imaging system was used to monitor tumor growth weekly, and mice were sacrificed 4 weeks after tumor implantation (a), measurements (photons/s) of tumor volume using live bioluminescence imaging at indicated times (b). (n = 5 mice per group). Values represent mean ± SEM. c Tumors from the two groups were dissected and photographed. (n = 5 mice per group). d, e Tumor volume (d) and weight (e) were measured on the last day of the experiment at autopsy. (n = 5 mice per group). f TKT activities in tumor lysates derived from orthotopically implanted CRC tumors were measured. (n = 5 mice per group). g R5P and nucleotide levels in orthotopically implanted tumors were measured by targeted metabolomics analysis. (n = 3 mice per group). h Western blotting of SIRT5, TKT, and ?H2AX in orthotopically implanted CRC tumors. GAPDH served as a loading control. Values indicate means ± SD in (d–g), compared by the two-sided Student’s t test. i–l HCT116 cells under different conditions were injected subcutaneously into nude mice. Tumors from different groups were dissected and photographed (i, j), and tumor volume and weight were measured (k, l). (n = 8 mice per group). m R5P and nucleotide levels in tumor lysates derived from subcutaneous xenograft tumors. (n = 3 mice per group). n Immunoblotting of SIRT5, TKT, phospho-p53 (Ser15), and ?H2AX proteins in subcutaneous xenograft tumor tissues under different treatments. o Representative TUNEL and ?H2AX staining of subcutaneous xenograft tumors at day 21. Scale bar, 50 µm. Values represent mean ± SD. One-way ANOVA with Tukey’s multiple comparisons test for (k–m). ns not significant. Source data are provided as a Source Data file.

Fig 3: SIRT5 activates TKT by mediating its demalonylation.a, b Immunofluorescent staining results for FLAG-SIRT5 WT/H158Y (in green) and TKT (in red). Yellow in the merged magnified images indicates co-localization. Scale bar, 5 µm (a). Fluorescence intensity of FLAG-SIRT5 WT/H158Y (green line) and TKT (red line) traced along the white line in HCT116 and LoVo cells using the line profiling function of ImageJ (b). This figure represents three independent experimental replicates with similar results. c Endogenous SIRT5 was immunoprecipitated with the anti-SIRT5 antibody, followed by Western blotting using an anti-TKT antibody in HCT116 and LoVo cells. The control comprised immunoprecipitation with IgG. d The interaction between FLAG-SIRT5 WT/H158Y and TKT in HCT116 and LoVo cells. e Malonylation (MalK) levels of exogenous TKT in HCT116 and LoVo cells expressing the control vector, SIRT5 WT, or SIRT5 H158Y. f The MalK levels of exogenous TKT in SIRT5-deficient HCT116 and LoVo cells were determined by Western blotting. g, h HA-tagged TKT proteins were purified using immunoprecipitation and incubated with different concentrations of malonyl-CoA (0, 1, and 2 mM) at 37 °C for 60 min. TKT activity was determined. Representative images (g) and quantification (h) of TKT activity. (n = 3 biologically independent experiments). i K281 of TKT is evolutionarily conserved across species. These sequences of TKT from humans to Gallus gallus were aligned. j HA-tagged TKT WT/K281R/K282R/K283R mutants were transfected into HCT116 cells, followed by treatment with SIRT5 siRNAs. TKT was immunoprecipitated and MalK levels were determined. k HCT116 cells expressing HA-tagged TKT WT/K281R mutant were treated with or without SIRT5 siRNAs. TKT activity was measured and normalized against protein levels. (n = 3 biologically independent experiments). Values in (h and k) represent the mean ± SD. P values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. ns, not significant. Source data are provided as a Source Data file.

Fig 4: TKT protects CRC cells from DNA damage, cell cycle arrest, and apoptosis after SIRT5 knockdown by the maintaining nucleotide pool.a Targeted metabolomics analysis of nucleotide levels in HCT116 cells stably expressing the control vector or SIRT5 WT treated with a control siRNA or siRNAs targeting TKT for 48 h. (n = 6 biologically independent experiments). b, c Representative immunofluorescence images of the EdU incorporation assay were captured in SIRT5-deficient HCT 116 and LoVo cells, after transfection with an empty vector or TKT plasmid (b). Scale bar, 5 µm. Data in (b) were quantified (c). (n = 3 biologically independent experiments). d, e Immunoblotting of ?-H2AX in HCT116 and LoVo cells stably expressing the control vector and SIRT5 WT, followed by treatment withTKT siRNAs (d) or OT (20 µM, e). 20 µM 5-FU was used as a DNA-damaging agent. f–i Flow cytometry was used to detect the effect of overexpressing TKT on the cell cycle (f) and apoptosis (h) in SIRT5-silenced CRC cells. The data in (f and h) were quantified and analyzed in (g) and (i) respectively. (n = 3 biologically independent experiments). j Western blotting showing that TKT overexpression inhibited the increased levels of cleaved caspase 3, caspase 8, caspase 9, PARP, and ?H2AX in SIRT5-silenced HCT116 and LoVo cells. Values in (a, c, g, and i) represent mean ± SEM. Experiments in (b–i) were performed three times independently with similar results. One-way ANOVA with Tukey’s multiple comparisons test was used for assessing significance. ns, not significant. Source data are provided as a Source Data file.

Fig 5: Schematic model showing that SIRT5 promotes the non-oxidative PPP by interacting with TKT, resulting in TKT demalonylation and activation.This maintains intracellular R5P and nucleotide levels, which supports CRC cell survival.