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. 2016 Dec 7;7:559. doi: 10.3389/fimmu.2016.00559

Figure 7.

Figure 7

Evidence for context-specific activities of hAAT. (A,B) S-NO-AAT induces pro-inflammatory effects on BMDM stimulation and tumor cell viability. (A) BMDMs (1 × 106 cells per well in triplicate) were pretreated with hAAT (2.5 mg/ml), S-NO-hAAT (2.5 mg/ml), or GSNO (27.5 μM) and after 24 h were cocultured with RMA cells (2.5 × 105 cells per well) with or without stimulation with IFNγ and IL-1β (both at 10 ng/ml). RMA cells were diluted 1:4 after 48 h, and after 72 h (i) cells were washed and stained for flow cytometric analysis, and (ii) supernatants were analyzed for NO levels. Mean ± SEM, *p < 0.05. Unpaired two-tailed Student’s t-test was employed to assess differences between groups. (B) RMA cells were seeded at 1 × 105 cells per well and treated with hAAT, S-NO-hAAT (both at 2.5 mg/ml), GSNO (27.5 μM), or doxorubicin (60 μM). (i) Cells were stained with trypan blue, and the population of viable cells was determined after 24 h. (ii) LDH release after 48 h. (iii) 7-AAD staining. Mean ± SEM, *p < 0.05, ***p < 0.001. Unpaired two-tailed Student’s t-test was employed to assess differences between groups. (C) Effect of ischemia–reperfusion on intra-tumor gene expression profile during hAAT treatment. C57BL/6 mice (n = 3 per group) were pretreated with hAAT (2 mg per animal, i.p.) 24 h prior to inoculation of B16-F10 melanoma cells (5 × 104 cells per mouse, i.f.p.). Mice were subjected to limb ischemia–reperfusion (3 h ischemia; 2 h reperfusion) on day 21 post-inoculation. Mice were then sacrificed, and total foot tissue surrounding the inoculation site was excised and processed for real-time QPCR gene expression analysis for indicated genes. Results are presented as fold-change from inoculated sham-operated mice. *p < 0.05, **p < 0.01 between hAAT+/+ and WT groups. Unpaired two-tailed Student’s t-test was employed to assess differences between groups.