Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Pharmacological induction of the hypoxia response pathway in Huh7 hepatoma cells limits proliferation but increases resilience under metabolic stress

doi: 10.1007/s00018-024-05361-6

Figure Lengend Snippet: Proliferation of Huh7 cells is inhibited by Complex III inhibitor antimycin, but is restored in the presence of pyruvate and Molidustat. A , B Huh7 cells were treated with DMSO alone, Molidustat (25 µM) and antimycin (1 µM) in the absence or presence of pyruvate (1 mM). After 72 h, cell counts were determined by Hoechst staining and quantification of the fluorescence signal ( A ) and ATP level in culture wells that reflects the number of viable cells was measured ( B ). Data were normalized to untreated control (None) without (left panels) or with pyruvate (right panels). Means ± SEM of four ( A ) and seven ( B ) experiments in triplicate. *p < 0.05, **p < 0.01; paired t-test. C Huh7 cells were treated for 72 h with Molidustat (25 µM) or DMSO alone (None), with or without 1 mM Pyruvate. Lactate accumulation in the extracellular medium was quantified and reported to control (None). Means ± SEM of five experiments in triplicate. **p < 0.01; paired t-test. D Huh7 cells were grown in culture medium supplemented with pyruvate (1 mM), LDHA inhibitor GSK2837808A at 3, 10, 30 and 90 µM, and DMSO (None), Molidustat (25 µM), antimycin (1 µM) or Molidustat plus antimycin. After 72 h, ATP level in culture wells was determined. For DMSO, Molidustat, antimycin or Molidustat plus antimycin, data were normalized to the control condition without GSK2837808A. Best fit curves as well as EC 50 values were calculated from the dose–response of GSK2837808A. Data correspond to the mean of six experiments in triplicate

Article Snippet: Molidustat was from Euromedex (France), Antimycin A and Uridine were from Sigma-Aldrich (France), Dipyridamole was from Bertin Bioreagent (France), and GSK2837808A was from MedChemExpress (Sweden).

Techniques: Staining, Fluorescence, Control

Journal: Cancer Science

Article Title: Neuromedin U regulates the anti‐tumor activity of CD8 + T cells and glycolysis of tumor cells in the tumor microenvironment of pancreatic ductal adenocarcinoma in an NMUR1‐dependent manner

doi: 10.1111/cas.16024

Figure Lengend Snippet: NMU regulates glycolysis in tumor tissues and affects CD8 + T‐cell function. (A) Real‐time PCR detected the relative expression of mRNAs for glycolysis‐related factors and Hif‐1α in tumor tissues. (B) Protein levels of key glycolytic factors in tumor tissues were detected using western blot. (C) The activities of PK and LDH in tumor tissue. (D) The concentration of lactic acid in tumor tissues. (E) Panc02 cells were stimulated with different concentrations of recombinant NMU for 24 h, and the expression of glycolysis‐related factors and Hif‐1α mRNAs were determined using real‐time PCR. (F, G) Panc02 cells were stimulated with 10 ng/mL of recombinant NMU for 24 h. The activities of PK and LDH in the cells (F) and the concentration of lactate in the supernatants of cultured cells (G). (H) Panc02 cells were stimulated with 10 ng/mL of recombinant NMU for 24 h in the presence or absence of 10 μM GSK2837808A. The concentration of lactic acid in the culture supernatants was determined. (I) CD8 + T cells were sorted from the spleens of tumor‐bearing mice and cultured in vitro with the supernatants from NMU‐treated or NMU + GSK2837808A‐treated Panc02 cells. Flow cytometry analyzed the percentages and absolute numbers of IFN‐γ + CD8 + T cells, perforin + CD8 + T cells, and granzyme B + CD8 + T cells. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 by Student t ‐test or one‐way ANOVA test. IFN‐γ, interferon‐γ; NMU, neuromedin U.

Article Snippet: In total, 1 × 10 5 of CD8 + T cells were sorted from the spleens of tumor‐bearing mice or tumor tissues using Miltenyi magnetically labeled beads (Miltenyi Biotec, Cologne, Germany), and cultured in a 24‐well plate coated with 1 μg/mL anti‐mouse CD3 ε and 2 μg/mL anti‐mouse CD28 ε (BioLegend) for 72 h. Then these activated CD8 + T cells were stimulated with 10 ng/mL of recombinant NMU in the presence or absence of 10 μM GSK2837808A (GlpBio GC13464, USA) for 24 h. The percentages of IFN‐γ‐, perforin‐ and granzyme B‐producing CD8 + T cells were collected by flow cytometry.

Techniques: Cell Function Assay, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Concentration Assay, Recombinant, Cell Culture, In Vitro, Flow Cytometry