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Figure 1: Mouse BM cells were cultured in medium containing GM-CSF (20 ng/mL) and IL-6 (20 ng/mL) for 6 days in the presence of 100 μM ALA or LA,ethanol was used as control. The proportion of MDSCs (Gr-1+CD11b+) was evaluated by flow cytometry. Left panels are from a single experiment, the right graph represents mean ± SEM of 6 samples pooled from 3 independent experiments. **p < 0.01: statistically different from control, unpaired t test was used.

Figure 2: BM cells from C57BL/6 mice were cultured in GM-CSF (20 ng/mL) for 6 days in the presence of 100 μM ALA or LA; ethanol was used as control.CD3+ T cells from BALB/c mice were stimulated with ConA, then cocultured with MDSCs isolated from the above cultured BM at different ratios for 3 days. T-cell proliferation was evaluated by CFSE dilution. Unstimulated T cells were used as a negative control. (A) The suppressive function of LA-induced MDSCs and control MDSCs. Left panels are from a single experiment; the right panels represent mean ± SEM of 6 samples pooled from 3 independent experiments. *p < 0.05, **p < 0.01: statistically significance compared with ConA-stimulated T cells.(B) The comparison in suppressive activity between PUFA-induced MDSCs and control MDSCs. Data represent mean ± SEM of 6 samples pooled from 3 independent experiments. *p < 0.05, **p < 0.01: compared with control MDSCs, unpaired t test was used.

Figure 3: Effects of different L-arginine related inhibitors on T-cell proliferation. CD3+ T cells from mouse spleen were stimulated with ConA for 3 days with treatments as indicated. T-cell proliferation was evaluated by CFSE dilution. Unstimulated T cells were used as a negative control. nor-NOHA(100 μM): arginase inhibitor; L-arginine(1 mM); L-NMMA (100 μM): iNOS inhibitor; NAC (1 mM): ROS inhibitor. Means ± SEM of 6 samples from 3 independent experiments are shown, unpaired t test was used.

Figure 4: Mice were fed diets enriched with ALA (LOD, linseed oil diet) or control diet (RD, regular diet) for 3 months. Gr1+CD11b+ cells were purified by flow cytometric sorting from BM and spleen, and the amounts of p-STAT3, STAT3, p47phox, S100A8/A9 proteins were determined by western blotting. Data are representative of 3 independent experiments.

Figure 5: Effect of PUFA on COX2 expression and the impact of COX2 inhibitor on PUFA-MDSC function. Mouse BM cells were cultured in GM-CSF (20 ng/mL) for 6 days with different treatments as indicated. NAC: ROS inhibitor, Nimesulide (Nim):COX2 inhibitor, ethanol was used as control. Gr1+CD11b+ cells were isolated from the cultured BM cells and subjected to the following experiments. (A) Expression of COX1 and COX2 was determined by qRT-PCR. (B) ROS level was determined by CM-H2DCFDA labeling.(C) Allogeneic MLR. MDSCs from the above cultures were cocultured with allogeneic CD3+ T cells stimulated with ConA, at 2:1 ratio for 3 days. T-cell proliferation was evaluated by CFSE dilution. Unstimulated T cells were used as a negative control. (A-C) Means ± SEM from 3 independent experiments are shown.

Table 1:Sequences of primers used in this study.

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