Fig 1: Kaplan–Meier curves for glioma patients with different levels of TAM biomarkers. Survival curves in patients that had tumors with positive CD68 (A), iNOS (B), and CD163 (C) were plotted. p < 0.05 was considered statistically significant.
Fig 2: iNOS-derived NO is required for C. tropicalis-induced glycolysis activation in MDSCs. A, B and E WT MDSCs were stimulated with heat-inactivated C. tropicalis (MOI = 1) in combination with or without specific iNOS inhibitor SMT (as indicated concentration) for 18 h. Cell lysates were analyzed by immunoblotting for the indicated proteins. C WT MDSCs were stimulated with heat-inactivated C. tropicalis (MOI = 5) in combination with or without specific iNOS inhibitor SMT (500 µM) for 24 h. Lactate secretion by MDSCs were determined by lactate assay kit. D MDSCs were stimulated with C. tropicalis (MOI = 2) in the presence or absence of specific iNOS inhibitor SMT (500 µM), then the ECAR level of these cells was measured by Agilent Seahorse XFe96 Analyzer. F and G WT MDSCs were treated with NO donor (as indicated concentration) for 12 h. Glycolytic enzymes mRNA expression were measured by q-PCR (F). Cell lysates were analyzed by immunoblotting for the indicated proteins (G). H WT MDSCs were treated with NO donor (250 µM and 500 µM) for 24 h. Lactate secretion by MDSCs were determined by lactate assay kit. I WT MDSCs were treated with NO donor (500 µM), then the ECAR level of these cells was measured by Agilent Seahorse XFe96 Analyzer. 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
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
Fig 4: Analysis for the diagnostic accuracy of using TAMs expression in gliomas by ROC curve. ROC analyses for the diagnostic accuracy of using CD68 (A), iNOS (B), and CD163 (C) expression were calculated. The AUC estimates evaluated by this approach are reported under the ROC association statistics section of data output.
Fig 5: Effects of Sal on PCAF expression, TUNEL staining, and iNOS expression in an in vivo mouse DMM model. (A, B) Immunofluorescence staining of PCAF expression in cartilage samples (scale bar: 50 µm). (C, D) TUNEL staining of cartilage samples (scale bar: 50 µm). (E, F) Immunohistochemical staining of iNOS expression in cartilage samples (scale bar: 50 µm). The data are presented as the means ± SDs (n = 6). Significant differences among different groups are indicated as ##P < 0.01 vs. the sham group; and **P < 0.01 vs. the DMM group.
Supplier Page from MilliporeSigma for Anti-INOS antibody produced in rabbit