Fig. 5
SIRT5 supports colorectal cancer growth by promoting glutamine metabolism via increased GLUD1 enzyme activity. a A diagram showing the enzymes involved in glutamine metabolism and the inhibitors used in this study. b Expression of GLUD1, GOT1/2, PSAT1, GPT2, and GLS (including kidney-type glutaminase (KGA isoform), and glutaminase C (GAC isoform)) upon SIRT5 knockdown. c GLUD1 enzyme activity was determined upon SIRT5 knockdown in HCT116 and LoVo cells. Left, representative images (n = 3). Right, quantification of GLUD1 activity. GLUD1 inhibitor epigallocatechin gallate (EGCG; 20 μM) was used as a positive control. d–f GLUD1 (d), GLS (e), and GOT2 (f) enzyme activities were determined in HCT116 and LoVo cells stably expressing the control vector, SIRT5 WT, or SIRT5 H158Y, respectively; n = 3. g Proliferation rate of HCT116 and LoVo cells stably expressing the control vector or SIRT5 WT plasmid in different culture conditions. DM glutamate (10 mM), DM α-KG (1 mM), and non-essential amino acids (NEAAs; 0.1 mM aspartate and asparagine) were added to glutamine-free medium, respectively; n = 4. h CCK-8 assays of HCT116 and LoVo cells stably expressing the control vector or SIRT5 WT treated with/without the specific GLUD1 siRNA; n = 5. i GLUD1 was knocked down in HCT116 and LoVo cells stably expressing the control vector or SIRT5 WT vector. Protein levels were assessed by western blotting. j Growth curves of HCT116 and LoVo cells after transfection of control siRNA (red line) and SIRT5 siRNAs (blue line) under the indicated conditions. Cells were cultured in standard media, and DM α-KG (1 mM) was added to media as indicated. The OD 450 was measured for 6 consecutive days using the CCK-8 assay; n = 5. k HCT116 and LoVo cells were transfected with control siRNA or SIRT5 siRNAs, and then cultured in standard media with DM α-KG (1 mM). The levels of cleaved PARP were detected by western blotting. Data in c–j are presented as the mean ± SD. P values were calculated by ANOVA. **P < 0.01, ***P < 0.001, N.S. = not significant for the indicated comparison
