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. 2026 Apr 8;12(15):eaec4368. doi: 10.1126/sciadv.aec4368

Fig. 4. SLC13A2 limits the TCA cycle and respiration to suppress tumor growth via citrate-mediated PKM2 acetylation and degradation.

Fig. 4.

(A) Relative abundance of TCA cycle and glycolysis metabolites in Hepa1-6 cells overexpressing SLC13A2 for 60 hours (n = 3). (B and C) Mitochondrial respiration (OCR) and glycolysis (ECAR) measured by Seahorse XF analysis. (D) Untargeted metabolomics of vector- or SLC13A2-transfected Hepa1-6 cells cultured with [U-13C6] glucose for 1 or 2 hours (n = 3). (E) Relative abundance of pyruvate (n = 3). (F) Cell viability/colony formation with the treatment of 500 μM pyruvate. *P < 0.05 versus vector without pyruvate; #P < 0.05 versus SLC13A2 without pyruvate. (G) Intracellular fractional labeling of citrate (m + 6) and acetyl-CoA (m + 2) in vector- or SLC13A2-transfected Hepa1-6 after 1 hour [U-13C6] citrate tracing (n = 3). (H and I) Relative abundance of oxaloacetate and extracellular/mitochondrial citrate levels (n = 3). (J) Cell viability after 72 hours ACLY inhibitor (ACLYi) treatment (50 μM). *P < 0.05 versus vector without ACLY inhibitor; #P < 0.05 versus SLC13A2 without ACLY inhibitor. (n = 4). (K) PKM2 subcellular protein levels after 60 hours transfection. (L) Co-IP assay showing the acetylation of PKM2. (M) PKM2 protein levels in SLC13A2-KD Hepa1-6 cells expressing WT/mutant PKM2, cultured for 60 hours. (N) Pyruvate kinase activity after 60 hours transfection (n = 3). (O) SLC13A2 and/or PKM2 overexpression efficiency (left) and cell growth curves measured at 0, 24, 72, and 120 hours (right). *P < 0.05 SLC13A2 versus vector; #P < 0.05 SLC13A2 + PKM2 versus SLC13A2. (P and Q) Pyruvate kinase activity (n = 10), pyruvate content (n = 10), and PKM2 protein levels (n = 6) in HTVi liver tissues with SLC13A2 LKO or OE. (R) NADH/NAD+ ratio in HTVi liver tissues with SLC13A2 overexpression (n = 6). Data are presented as means ± SEM. *P < 0.05, two-tailed unpaired Student’s t test [(A), (D), (E), (G) to (I), (N), and (P) to (R)]; two-way analysis of variance (ANOVA), followed by Bonferroni multiple comparisons [(F), (J), and (O)]. h, hour; IB, immunoblot; NC, Negative Control.