Journal: Frontiers in Immunology

Article Title: Extracellular CIRP-Impaired Rab26 Restrains EPOR-Mediated Macrophage Polarization in Acute Lung Injury

doi: 10.3389/fimmu.2021.768435

Figure Lengend Snippet: Rab26 deficiency reduces EPOR signaling and restrains macrophage polarization. A qPCR assay was conducted to evaluate the mRNA expression of EPOR (A) and PPARγ (B) in WT BMDMs treated with rhEPO (20 IU/ml) for the indicated time (n = 3). (C) The MFIs of cell surface EPOR in BMDMs treated with rhEPO (20 IU/ml) for the indicated times were tested by FACS (n = 3). * P < 0.05, ** P < 0.01 vs. 0 h. (D) The MFIs of CD80 and CD86 were evaluated by FACS in WT and Rab26 -/- BMDMs treated with or without eCIRP (1 µg/ml) or rhEPO (20 IU/ml) for 24 h (n = 3). (E) A qPCR assay was conducted to evaluate the mRNA expression of TNF-α, IL-6, and IL-1β in WT and Rab26 -/- BMDMs treated with eCIRP (1 µg/ml) and rhEPO (20 IU/ml) for 24 h (n = 3). (F) Localization of EPOR in WT and Rab26 -/- BMDMs. WT and Rab26 -/- BMDMs were stained with an anti-EPOR antibody (1:100 dilution) and Alexa Fluor 488-labeled secondary antibody (1:200 dilution) (green), and nuclei were stained with DAPI (blue). Representative confocal images of the surface and intracellular expression of EPOR are shown. Scale bar: 10 µm. 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) . EPOR, erythropoietin receptor; PCR, polymerase chain reaction; PPAR, peroxisome proliferator-activated receptor; BMDM, bone marrow derived macrophage; EPO, erythropoietin; MFI, mean fluorescence intensity; FACS, fluorescence activated cell sorter; eCIRP, extracellular cold-inducible RNA-binding protein.

Article Snippet: Reagents were as follows: LPS from Escherichia coli O111:B4 (Sigma-Aldrich, #L4391), LPS from Escherichia coli 055:B5 (Sigma-Aldrich, #L2880), human CIRBP/CIRP (Sino Biological, #14578-H07E), rhEPO (Sunshine Pharmaceutical, Shenyang, China), cell dissociation buffer (Gibco, #13150016), PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, #23225), TRIzol Reagent (Sigma-Aldrich, #T9424), cOmpleteTM EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich, #04693159001), GoScriptTM Reverse Transcription System (Promega, #A2800), GoTaq ® qPCR Master Mix (Promega, #A6001), M-PER Protein Extraction Reagent (Thermo Fisher Scientific, #78510), PageRuler Prestained Protein Ladder (Thermo Fisher Scientific, #26616), Immobilon Western Chemiluminescent HRP Substrate (Millipore, #WBKLS0500), LEGENDplexTM Multi-Analyte Flow Assay Kit (BioLegend, #740740), Immunofluorescence Application Solutions Kit (CST, #12727), Anti-fade Reagent with DAPI (Coolaber, #SL 1841), and PE Annexin V Apoptosis Detection Kit (BD, #559763).

Techniques: Expressing, Staining, Labeling, Polymerase Chain Reaction, Derivative Assay, Fluorescence, RNA Binding Assay