Journal: Cells

Article Title: ACLY Nuclear Translocation in Human Macrophages Drives Proinflammatory Gene Expression by NF-κB Acetylation

doi: 10.3390/cells10112962

Figure Lengend Snippet: LPS induced ACLY acetylation and, in turn, its nuclear translocation. Human PBMC-derived macrophages were triggered by LPS, and ( a ) nuclei were isolated by cell fractionation. In nuclear and cytoplasm fractions, ACLY, β-actin, and lamin proteins were detected by specific antibodies. ( b ) Nuclear ACLY activity. ( c ) Time course of ACLY nuclear translocation by immunocytochemistry (ICC) using anti-ACLY and DAPI nuclear staining. ( d ) Immunoprecipitated with an antibody directed to acetylated lysine and analyzed by western blot with an anti-ACLY antibody. ( e ) In macrophages triggered by LPS, ACLY was immunoprecipitated with a specific antibody, and then an antibody against acetylated lysine (Ac. Lys.) was used in western blotting experiments. ( f ) A 3D model of ACLY protein based on 6hxh.pdb is reported in a green cartoon representation and in a complex with citrate (blue spheres), ADP (yellow spheres), Mg (white spheres), and CoA (orange spheres). K662 and K665 acetylated lysines are reported in red sticks. (Acetyl groups added by PyMOL on K662 and K665 are reported in white sticks) ( g ) iBMDM cells were transfected with a construct overexpressing the ACLY wild type (Pwt) and a construct overexpressing a double Ala mutant (Pmut) and used for isolating nuclei. In nuclear and cytoplasm fractions, ACLY, β-actin, and lamin proteins were detected by specific antibodies. ( h ) iBMDM cells, transfected as in ( g ), were used for ICC analysis with anti-ACLY and DAPI nuclear staining. ( i ) iBMDM cells, transfected as in ( g ), were used for ICC analysis with an anti-DDDDK tag and DAPI nuclear staining. In ( a , b ), IgG is the negative control. In ( b ), data are representative of 3 independent experiments and are presented as means ± SD (error bars). Statistical significance of the differences was evaluated by using one-way ANOVA followed by Dunnett’s multiple comparison test (*** p < 0.001). Western blotting and ICC data presented are representative of at least 3 independent experiments. In ( a , g ), protein levels are quantified against β-actin or lamin. In ( a , d , e , g ), they are normalized versus the mean of proteins in untreated cells (C) and reported under each image. Quantitation of ACLY in ( c , h , i ) ICC images was normalized to a DAPI nuclear stain.

Article Snippet: The day after, an Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (A-11008, Thermo Fisher Scientific) was used while Fluoroshield Mounting Medium with DAPI (ab104139, Abcam) was employed to preserve the fluorescence and as a counterstain for DNA.

Techniques: Translocation Assay, Derivative Assay, Isolation, Cell Fractionation, Activity Assay, Immunocytochemistry, Staining, Immunoprecipitation, Western Blot, Transfection, Construct, Mutagenesis, Negative Control, Comparison, Quantitation Assay

Journal: Cells

Article Title: ACLY Nuclear Translocation in Human Macrophages Drives Proinflammatory Gene Expression by NF-κB Acetylation

doi: 10.3390/cells10112962

Figure Lengend Snippet: LTA and sepsis in the early hyperinflammatory phase triggered ACLY-mediated NF-κB acetylation. Human PBMC-derived macrophages were triggered by LTA, and ( a ) western blot experiments were performed to evaluate ACLY protein. ( b ) Nuclei were isolated by cell fractionation. In nuclear and cytoplasm fractions, ACLY, β-actin, and lamin proteins were detected by specific antibodies. ( c ) Time course of ACLY nuclear translocation by immunocytochemistry (ICC) using anti-ACLY and DAPI nuclear staining in iBMDM cells treated with LTA. ( d ) In human PBMC-derived macrophages triggered by LTA, with or without SB, NF-κB (p65) was immunoprecipitated with a specific antibody and then analyzed by western blotting with an anti-NF-κB p65 (acetyl K310) antibody. ( e , f ) Macrophages differentiated from PBMCs of patients with sepsis in the early hyperinflammatory phase and age-matched healthy controls were used to quantify ACLY protein by western blot experiments ( e ) and to immunoprecipitate NF-κB (p65) with a specific antibody and then to analyze by western blotting with an anti-NF-κB p65 (acetyl K310) antibody. ( f ) Western blotting data presented are representative of at least 3 independent experiments. In ( a , b , e ), protein levels are quantified against β-actin. In ( a , b , d ), the mean of the protein values was normalized versus the mean of the proteins in untreated cells (C), and the results are reported under each image. In ( c ), quantitation of ACLY in ICC experiments was normalized to the DAPI nuclear stain. In ( e , f ), statistical significance of the differences between the ACLY amount in the controls and sepsis samples evaluated by using a Mann–Whitney U test (** p < 0.01) is depicted in dot plots (right panels).

Article Snippet: The day after, an Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (A-11008, Thermo Fisher Scientific) was used while Fluoroshield Mounting Medium with DAPI (ab104139, Abcam) was employed to preserve the fluorescence and as a counterstain for DNA.

Techniques: Derivative Assay, Western Blot, Isolation, Cell Fractionation, Translocation Assay, Immunocytochemistry, Staining, Immunoprecipitation, Quantitation Assay, MANN-WHITNEY

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