Fig. 3

Glycolysis mediates C. tropicalis-enhanced immunosuppressive function of MDSCs. A WT MDSCs were stimulated with heat-inactivated C. tropicalis (MOI = 1) in combination with or without glycolysis inhibitor 2-DG (1 mM) for 24 h. 7AAD viability was determined by flow cytometry. B and C WT MDSCs were stimulated with heat-inactivated C. tropicalis (MOI = 1) in the presence or absence of glycolysis inhibitor 2-DG (as indicated concentration) for 12 or 18 h. iNOS, COX2, NOX2 mRNA expression were measured by q-PCR (B). Cell lysates were analyzed by immunoblotting for iNOS, COX2, NOX2 (C). D WT MDSCs were stimulated with heat-inactivated C. tropicalis (MOI = 5) in the presence or absence of glycolysis inhibitor 2-DG (1 mM) for 48 h. NO concentration in culture supernatants was measured by NO assay kit. E-G WT MDSCs were pretransfected with HK2 siRNA for 24 h prior to stimulation with heat-inactivated C. tropicalis (MOI = 2) for 12 or 24 h. Nos2, Ptgs2 and Cybb mRNA expression were measured by qPCR (F). Cell lysates were analyzed by immunoblotting for HK2 (E), iNOS, COX2 and NOX2 (G). H and I The suppressive effect of MDSCs on the proliferation of CD8⁺ T cells was analyzed by flow cytometry. The results shown here are expressed as the mean ± SEM. Each panel is a representative experiment of at least three independent biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001. The following statistical analyses were performed: unpaired Student’s t-test or one-way ANOVA where appropriate