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. 2022 Aug 9;13:938944. doi: 10.3389/fimmu.2022.938944

Figure 3.

Figure 3

EPO mediated re-programming of endotoxin-tolerant macrophages through PI3K/AKT pathway via upregulation of IRAK3 and WDR5 (A, B): In vitro cultured RAW264.7 macrophages were incubated with PBS, rhEPO (40 IU/ml), and rhEPO (40 IU/ml) + LY294002 (20 μM) (A) or PBS, rhEPO (40 IU/ml), and rhEPO (40 IU/ml) + MK2206 (5 μM) (B) the in presence of LPS (100 ng/ml) for 24 h, and then cells were washed with PBS twice followed by a secondary LPS stimulation (10 ng/ml) for 6 h; gene expression was measured by qRT-PCR (n = 3). (C): In vitro cultured RAW264.7 macrophages were incubated with PBS, rhEPO (40 IU/ml), or rhEPO (40 IU/ml) + MK2206 (5 μM) in the presence of LPS (100 ng/ml) for 24 h, and then gene expression of Irak3 and Wdr5 was measured by qRT-PCR (n = 3). (D–F): RAW264.7 macrophages were incubated with PBS, rhEPO (40 IU/ml), and rhEPO (40 IU/ml) + Irak3 siRNA (50 nM) (D); PBS, rhEPO (40 IU/ml), and rhEPO (40 IU/ml) + WDR5-0103 (500 nM) (E); or PBS, rhEPO (40 IU/ml), and rhEPO (40 IU/ml) + 5-AZA (5 μM) (F) in the presence of LPS (100 ng/ml) for 24 h, and then cells were washed with PBS twice followed by a secondary LPS stimulation (10 ng/ml) for 6 h; gene expression was measured by qRT-PCR (n = 3). Data are representative of three independent experiments. Results were expressed as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. n.s., not statistically significant. Statistics: two-way ANOVA with Tukey’s post-hoc test for multiple comparisons. "n.s." stands for "not statistically significant“.