Figure 4.

Myeloid EPOR signaling is essential for macrophage polarization. (A) A qPCR assay was conducted to evaluate the mRNA expression of EPOR and PPARγ after WT BMDMs were treated with eCIRP for the indicated times (n = 3). (B) The MFIs of cell surface EPOR in BMDMs treated with eCIRP for the indicated times were tested by FACS (n = 3). **P < 0.01 vs. 0 h group. (C) The percentage of EPOR⁺ cells in WT and EPOR cKO BMDMs treated with or without eCIRP for 24 h (n = 3). (D) The MFIs of CD80 and CD86 in WT and EPOR cKO BMDMs were measured by FACS with or without eCIRP (1 µg/ml) and rhEPO (20 IU/ml) administration (n = 3). (E) A qPCR assay was conducted to evaluate the mRNA expression of TNF-α, IL-6, and IL-1β in WT and EPOR cKO BMDMs with or without eCIRP and rhEPO (20 IU/ml) administration (n = 3). (F) The supernatant concentrations of TNF-α, IL-6, and IL-10 in BMDMs were measured with or without eCIRP (1 µg/ml) and rhEPO (20 IU/ml) administration (n = 3). (G) Western blot analysis of PPARγ and β-actin protein expression was conducted after WT and EPOR cKO BMDMs were treated with eCIRP for 24 h (n = 3). Data are representative of at least two independent experiments. Results were expressed as mean ± SD. n.s., not statistically significant. *P < 0.05, **P < 0.01. Statistics: One-way ANOVA with Tukey’s post-hoc test for multiple comparisons (A–G). EPOR, erythropoietin receptor; PCR, Polymerase Chain Reaction; PPAR, peroxisome proliferator-activated receptor; BMDM, bone marrow derived macrophage; eCIRP, extracellular cold-inducible RNA-binding protein; WT, wild type; EPO, erythropoietin; MFI, mean fluorescence intensity; FACS, fluorescence activated cell sorter.