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. 2021 Dec 1;12:768435. doi: 10.3389/fimmu.2021.768435

Figure 6.

Figure 6

Rab26 is critical for macrophage polarization. (A) Macrophages in BALF from WT-ALI mice (3 mg/kg LPS i.t.) were isolated by magnetic-activated cell sorting with F4/80 labeled, and qPCR analysis of the Rab26 mRNA expression in BALF macrophages was tested at the indicated times (days 0, 1, 2, 3, and 5) (n = 3). *P <0.05, **P <0.01 vs. the day-0 group. A qPCR assay was conducted to evaluate the mRNA expression of Rab26, (B) and Western blot analysis of Rab26 and β-actin protein expression (C) was conducted after WT BMDMs were treated with eCIRP for the indicated times (n = 3). *P < 0.05, **P < 0.01 vs. 0 h group. The membrane CD80 MFI of BMDMs (D) and the percentage of CD206+ BMDMs (E) were measured by FACS with or without eCIRP (1 µg/ml) administration for 24 h (n = 3). (F) A qPCR assay was conducted to evaluate the mRNA expression of EPOR and PPARγ in WT and Rab26-/- BMDMs (n = 3). (G) The membrane EPOR of WT and Rab26-/- BMDMs was evaluated by FACS (n = 3). *P < 0.05, **P < 0.01 vs. WT group. 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–E) or unpaired two-tailed Student’s t-test (F, G). EPOR, erythropoietin receptor; BALF, bronchoalveolar lavage fluid; ALI, acute lung injury; 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.