Journal: Molecular Biomedicine

Article Title: Lactate-driven pyrimidine synthesis promotes ferroptosis resistance in hepatocellular carcinoma

doi: 10.1186/s43556-026-00450-3

Figure Lengend Snippet: Effects of pyrimidine biosynthesis-driven pre-rRNA synthesis on lactate-induced ECM production. a , b Effects of 24 h exposure to AR-C155858 (2 µM, a) or BRQ (0.1 µM, b) on puromycin incorporation in LX-2 cells cultured in the presence or absence of lactate (10 mM). c Immunofluorescence staining for puromycin (1 µg/mL) in LX-2 cells showing the effects of BRQ (0.1 µM, 24 h) in CM from Huh7 cells (left) or in the presence or absence of lactate (10 mM, 24 h, right). d Pull-down assay showing the effects of BRQ (0.1 µM, 24 h) on COL1A1 and FN1 protein levels in LX-2 cells cultured in the presence or absence of lactate (10 mM, 24 h). e UTP levels in LX-2 cells treated with BRQ (0.1 µM, 24 h) in CM from Huh7 cells (left) or in the presence or absence of lactate (10 mM, 24 h, right) ( n = 3). f Effects of BRQ or uridine on COL1A1 and FN1 protein levels in LX-2 cells. LX-2 cells were treated with BRQ (0.1 µM) or uridine (5, 20, or 50 µM) in the absence or presence of lactate (10 mM) for 24 h (left panel). LX-2 cells cultured in Huh7-derived CM were treated with BRQ (0.1 µM) in the absence or presence of uridine (50 µM) for 24 h (right panel). g Immunofluorescence staining of Y10B in LX-2 cells showing the effects of BRQ (0.1 µM) or uridine (50 µM) exposure for 24 h in CM from Huh7 cells (left) or in the presence or absence of lactate (10 mM, right). h Relative expression of 5′ ETS in LX-2 cells treated with BRQ (0.1 µM) or uridine (50 µM) for 24 h in CM from Huh7 cells (left) or in the presence or absence of lactate (10 mM, right) ( n = 3). i Effects of CX-5461 (0.1 µM, 24 h) on COL1A1 and FN1 protein levels in LX-2 cells cultured in CM from Huh7 cells (left) or in the presence or absence of lactate (10 mM, 24 h, right). j Effects of MCT1 - or DHODH -targeting siRNA on COL1A1 (red) and FN1 (green) immunofluorescence staining in LX-2 cells co-cultured with Huh7 cells and treated with uridine (50 µM, 24 h). k Schematic model illustrating lactate-driven mTORC1 activation and its role in ECM production via the pre-rRNA synthesis pathway in HSCs. Created with BioRender.com, agreement number: MP29JVSCPU. Scale bar: 75 µm. Data are presented as the mean ± SEM from at least three independent experiments. n.s., Not significant; ** p < 0.01; *** p < 0.001. BRQ, Brequinar

Article Snippet: LX-2 cells were treated with CM, sodium L-lactate (10 mM; Sigma), AR–C 155858 (2 μM; MedChemExpress, Monmouth Junction, NJ, USA), rapamycin (20 nM; Sigma), PALA (150 μM; MedChemExpress), BRQ (0.1 μM; Selleckchem, Houston, TX, USA), uridine (5, 10, 50 μM; Sigma), cytidine (50 μM, Sigma), mitoQH 2 (0.1 μM; Cayman, Ann Arbor, MI, USA), and CX-5461 (0.1 μM; MedChemExpress) for 24 h. Huh7 and PLC/PRF/5 cells were treated with IKE (1 μM; Selleckchem) and sorafenib (10 μM; Selleckchem) for 6 h, and ferrostatin-1 (1 μM, Sigma), Z-VAD-FMK (20 μM; Selleckchem), necrostatin-1 (20 μM; MedChemExpress), and MGH-CP1 (2 μM, MedChemExpress) were applied for 24 h. For collagen and fibronectin stimulation, cells were serum-starved for 10 h and then treated with collagen I (30 μg/mL; Corning, NY, USA) or fibronectin (30 μg/mL; R&D Systems, MN, USA) for 24 h in the presence or absence of verteporfin (1 μM; MedChemExpress).

Techniques: Cell Culture, Immunofluorescence, Staining, Pull Down Assay, Derivative Assay, Expressing, Activation Assay