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

Journal: International Journal of Biological Sciences

Article Title: Extracellular Vesicles (EVs) from Lung Adenocarcinoma Cells Promote Human Umbilical Vein Endothelial Cell (HUVEC) Angiogenesis through Yes Kinase-associated Protein (YAP) Transport

doi: 10.7150/ijbs.31605

Figure Lengend Snippet: YAP in H1975 cells is transferred to HUVECs via EVs. ( A ) ELISA was used to measure the level of YAP in the H1975 supernatant, with pure DMEM used as a control. ( B ) We observed EVs that were isolated from H1975 cell supernatants by TEM. The scale bar represents 200 nm. ( C ) The size distribution of the EVs was examined using NanoSight NS300. ( D ) The EV marker proteins CD81, TSG101, Alix and CD63 were detected by western blotting. ( E ) YAP in the EVs purified from H1975 cell supernatants was detected by western blotting. DMEM was used as a control. ( F ) The level of YAP in HUVECs that were cocultured with EVs at different concentrations was detected by western blotting. GAPDH was used as the internal reference. ( G ) The mRNA level of YAP in HUVECs that were cocultured with H1975 supernatant-purified EVs at different concentrations was detected by real-time quantitative PCR. GAPDH was used as an internal reference. ( H ) HUVECs were cocultured with the H1975 supernatant and EV-free supernatant. The YAP level in HUVECs was detected by western blotting, and GAPDH was used as the internal reference. ( I ) EVs from H1975 cells were labeled with CM-Dil (red) and added to DIO labeled HUVECs for 2 h, and were observed by confocal microscopy. DAPI staining was used to visualize the nuclear areas. The scale bar represents 20 μm. * P < 0.05; ** P < 0.01 was considered statistically significant.

Article Snippet: The antibodies to human CD81, Alix, Tsg101 were purchased from Abcam.

Techniques: Enzyme-linked Immunosorbent Assay, Isolation, Marker, Western Blot, Purification, Real-time Polymerase Chain Reaction, Labeling, Confocal Microscopy, Staining

Journal: The Journal of Biological Chemistry

Article Title: Characterization of the Properties of a Novel Mutation in VAPB in Familial Amyotrophic Lateral Sclerosis *

doi: 10.1074/jbc.M110.161398

Figure Lengend Snippet: Protein properties are modified by T46I. A , COS-7 cells were transfected with the indicated plasmids and fixed 48 h after transfection. The distribution of VAPB protein is indicated by GFP ( green ), and nuclei are shown with DAPI staining ( blue ). Scale bar , 20 μm. B , NSC-34 cells were harvested 48 h after transfection with empty GFP vector or GFP VAPBs. The separation of Triton X-100-soluble and insoluble fraction was carried out as described by Kanekura et al. . The fractions were blotted with anti-GFP antibody. The densitometry measurements of bands for soluble and insoluble VAPB are shown in C . Values are means ± S.E. ( error bars ) for three separate experiments.

Article Snippet: Antibodies used were rabbit polyclonal anti-GFP (1:3000; Abcam), rabbit polyclonal anti-ubiquitin (1:200; Dako), rabbit polyclonal anti-phospho-eIF2α (1:1000; Cell Signaling), anti-β-actin (1:25000; Sigma), peroxidase-labeled goat anti-rabbit IgG (1:1000; Vector), and Alexa Fluor 594 goat anti-rabbit IgG (1:2000; Invitrogen).

Techniques: Modification, Transfection, Staining, Plasmid Preparation