Journal: The FASEB Journal

Article Title: Extracellular vesicles extracted from young donor serum attenuate inflammaging via partially rejuvenating aged T-cell immunotolerance

doi: 10.1096/fj.201800059R

Figure Lengend Snippet: Characteristics of serum-extracted EVs. A) Comparison of the sizes of EVs extracted from young murine serum by the ExoQuick reagent pretreated with and without using 0.2-μm filters. B) Morphology of EVs from young murine serum used in this project for rejuvenation of inflammaging, photographed by atomic force microscopy (AFM). C, D) Different miRNA expression profiles in heatmap (C) and quantified summary (D) of EVs from young vs. old murine serum, analyzed by murine miRNA microarray with Mus musculus miRBase version-21 arrays that contained 1900 unique mature miRNA probes (miRNA microarray service via LC Sciences).

Article Snippet: Then, 1–3 μg of total RNAs for each sample were used for customized miRNA microarray service from LC Sciences (Houston, TX, USA).

Techniques: Comparison, Microscopy, Expressing, Microarray

Journal: Gene

Article Title: Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures

doi: 10.1016/j.gene.2012.09.093

Figure Lengend Snippet: Distinct miRNA profiles of mK-ras-LE cells in 2-D and rBM 3-D cultures. A) mK-ras-LE cells were grown in 2-D and rBM 3-D culture. Morphology of the cells was visualized using fluorescent staining for F-actin. The images of rBM 3-D culture were captured at the central planes of cell clusters using a confocal fluorescent microscope on day 12 post-seeding. The images were captured at 600x magnification. Representative images of each culture condition were displayed. B) Total cell RNA was extracted from mK-ras-LE cells in 2-D and rBM 3-D cultures. miRNA expression profiles were compared between the two culture conditions using miRNA microarrays. A heatmap of the significantly differentially expressed miRNAs was generated using geWorkbench suite (n = 3, P < 0.01, FDR < 0.05). C) The expression of miR-17, miR-92b, and miR-21 was measured using qRT-PCR in total cell RNA collected from 2-D and rBM 3-D cultures of mK-ras-LE cells. A fold change of each miRNA of interest was obtained by setting the values of each miRNA in 2-D culture to one and normalization to the values of U6. D) Similar to part C except that the expression of miR-200a was compared between 2-D and rBM 3-D cultures. E) The culture conditions were similar to part A. The cell lysates were collected from the cell colonies extracted from the culture. The protein levels of ZEB2, PTEN, and -actin were determined using immunoblots. The data were presented in averages and standard deviations obtained from three independent experiments. and indicated a P value < 0.05 and 0.01, respectively.

Article Snippet: The miRNA expression profiling service was provided by LC Sciences (Houston, TX) using μParaflo® technology and proprietary probe hybridization (Cy3 and Cy5 dendrimer dyes) that enables highly sensitive and specific direct detection of mouse miRNAs.

Techniques: Staining, Microscopy, Expressing, Generated, Quantitative RT-PCR, Western Blot

Journal: Gene

Article Title: Comparative profiling of miRNA expression of lung adenocarcinoma cells in two-dimensional and three-dimensional cultures

doi: 10.1016/j.gene.2012.09.093

Figure Lengend Snippet: miRNA expression associated with aincar morphogenesis. A) A549 and A549LC cells were cultured in 2-D (A1 & A2) and rBM 3-D (A3 & A4) cultures. Morphology was recorded using an inverse phase contrast microscope. Polarized monolayer of the cells and the central lumen were pointed at by white arrows. B) The expression of miR-17, miR-92, and miR-21 was measured using qRT-PCR in total cell RNA collected from 2-D and rBM 3-D of A549 and A549LC cells. A fold change of each miRNA was obtained by setting the values of each miRNA from A549 cells in 2-D culture to one and normalization to the values of U6. C) Similar to part A except that the expression of miR-200a was compared across the groups. D) The culture conditions were similar to part A. The RNA levels of pri-miR-21 and VMP1 were measured in A549 cells using qRT-PCR. A fold change of each RNA was obtained by setting the values of RNA from A549 cells in 2-D culture to one and normalization to the values of 36B4. The data were presented in averages and standard deviations obtained from three independent experiments. , , and indicated a P value < 0.05, 0.01, and 0.001, respectively.

Article Snippet: The miRNA expression profiling service was provided by LC Sciences (Houston, TX) using μParaflo® technology and proprietary probe hybridization (Cy3 and Cy5 dendrimer dyes) that enables highly sensitive and specific direct detection of mouse miRNAs.

Techniques: Expressing, Cell Culture, Microscopy, Quantitative RT-PCR

Journal: Circulation. Heart failure

Article Title: Differential microRNA-21 and microRNA-221 upregulation in the biventricular failing heart reveals distinct stress responses of right versus left ventricular fibroblasts

doi: 10.1161/CIRCHEARTFAILURE.119.006426

Figure Lengend Snippet: (A)Volcano plot of miRNA expression in RV-HF vs RV-Ctrl. Blue dots, miRNAs that were differentially expressed at P<0.10. Labeled dots, miRNAs that were differentially expressed at a minimum 2-fold change in either direction (n=3 per group). (B)Heat maps, Venn diagram, and summary bar graph of differentially expressed miRNAs. Orange font, differentially expressed in RV-HF vs RV-Ctrl and in LV-HF vs LV-Ctrl but not statistically significantly different in RV-HF vs LV-HF. Blue font, differentially expressed in RV-HF vs RV-Ctrl and in RV-HF vs LV-HF, but not statistically significantly different in LV-HF vs LV-Ctrl. Purple font, differentially expressed across all three comparisons: LV-HF vs LV-Ctrl, RV-HF vs RV-Ctrl, and RV-HF vs LV-HF. *P<0.05 vs respective LV-HF/LV-Ctrl. (C)Quantitative RT-PCR analysis of miR-21 and miR-221 in ventricular tissue, n= 6 per group. *P<0.01 vs respective Ctrl; #P<0.01 vs LV HF. (D)Cyclic overstretch and/or aldosterone induced a marked increase in miR-21 (*P<0.01 vs unstimulated) and (E)miR-221 (*P<0.05 vs unstimulated) only in RV fibroblasts. (F)Inhibition of miR-21/−221 attenuated proliferation in RV but not LV fibroblasts. n= 4 per experimental condition. *P<0.05 vs respective LV, #P<0.05 vs RV without antimir, analyzed by ANOVA on Ranks.

Article Snippet: miRNA microarray data was analyzed for differential miRNA expression between pre-specified groups (RV-HF vs. RV-Ctrl, LV-HF vs. LV-Ctrl, and RV-HF vs. LV-HF) by two-tailed Student’s t-test using GraphPad Software (Prism 7.0).

Techniques: Expressing, Labeling, Quantitative RT-PCR, Inhibition

Journal: PLoS ONE

Article Title: Profiling Circulating MicroRNA Expression in Experimental Sepsis Using Cecal Ligation and Puncture

doi: 10.1371/journal.pone.0077936

Figure Lengend Snippet: Clustering of differentially expressed circulating microRNAs in the whole blood of C567BL/6 mice at 4, 8, and 24 h after cecal ligation and puncture (A) and subcutaneous bacteria injection with 1×10 8 E. coli. or S. aureus (B).

Article Snippet: Mouse genome-wide miRNA microarray analysis and statistical analysis was performed by Phalanx Biotech.

Techniques: Ligation, Bacteria, Injection

Journal: PLoS ONE

Article Title: Profiling Circulating MicroRNA Expression in Experimental Sepsis Using Cecal Ligation and Puncture

doi: 10.1371/journal.pone.0077936

Figure Lengend Snippet: Venn diagram showing the common and specific up-regulated miRNAs in the cecal ligation and puncture experiment without and after subcutaneous injection of gram-negative or gram-positive bacteria.

Article Snippet: Mouse genome-wide miRNA microarray analysis and statistical analysis was performed by Phalanx Biotech.

Techniques: Ligation, Injection, Bacteria

Journal: Signal Transduction and Targeted Therapy

Article Title: Natural killer cell-derived exosomal miR-1249-3p attenuates insulin resistance and inflammation in mouse models of type 2 diabetes

doi: 10.1038/s41392-021-00805-y

Figure Lengend Snippet: NK-derived exosomes from lean mice attenuate obesity-induced insulin resistance. a Fasting blood glucose of NCD and HFD mice after group feeding. b – d OGTT ( b ), ITT ( c ), and HOMA-IR index ( d ) of HFD mice and NCD mice ( n = 48) were recorded at 42 days after group feeding. HOMA-IR = Fasting blood glucose value × fasting serum insulin value/22.5. e Transmission electron micrographs of NK-derived exosomes. Scale bar, 100 nm. f NanoSight particle tracking analysis showing the particle size of NK-derived exosomes isolated from NCD and HFD mice. g Western blot assays of exosomal markers TSG101, HSP70, CD63, and CD9. h – k After blank liposomes, NCD-Exos, and HFD-Exos were separately injected into NCD or HFD mice via tail vein, fasting blood glucose ( h ), OGTT ( i ), ITT ( j ), and HOMA-IR ( k ) were assessed in each group. G1: HFD mice treated with blank liposomes ( n = 3); G2: HFD mice treated with HFD-Exos ( n = 3); G3: HFD mice treated with NCD-Exos ( n = 3); G4: NCD mice treated with blank liposomes ( n = 3); G5: NCD mice treated with HFD-Exos ( n = 3); and G6: NCD mice treated with NCD-Exos ( n = 3). l After NCD-Exos labeled with PKH26 were transferred into recipient mice, fluorescence images of the liver, islets, SATs, VATs, and skeletal muscles were observed by in vitro imaging system. Scale bar, 5 mm. m The weight of VATs from HFD mice treated with NCD-Exos, HFD-Exos, or blank liposomes. n Liver triglyceride content of HFD mice treated with NCD-Exos, HFD-Exos, or blank liposomes. o , p ELISA assays of IL-6, IL-1β, and TNF-α expression in VATs and livers of HFD mice treated with NCD-Exos, HFD-Exos, or blank liposomes. Experiments were performed at least in triplicate, and the results are shown as the mean ± s.d. Student’s t-test was used to analyze the data. (* P < 0.05; ** P < 0.01; *** P < 0.001)

Article Snippet: Exosomal miRNA microarray analysis was performed at CapitalBio Technology, Inc. (Beijing, China) using Agilent Mouse miRNA 8 × 60K V21.0 Microarray (Agilent Technologies, California, USA).

Techniques: Derivative Assay, Transmission Assay, Isolation, Western Blot, Liposomes, Injection, Labeling, Fluorescence, Muscles, In Vitro, Imaging, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Natural killer cell-derived exosomal miR-1249-3p attenuates insulin resistance and inflammation in mouse models of type 2 diabetes

doi: 10.1038/s41392-021-00805-y

Figure Lengend Snippet: NK-derived exosomal miR-1249-3p mediates cellular insulin sensitivity and inflammation. a Microarray analysis of significantly expressed exosomal miRNAs between NCD-Exos and HFD-Exos was presented in a heatmap. b qRT-PCR assay of miR-1249-3p expression in splenic NK cells, NK-derived exosomes, and circulating exosomes from NCD or HFD mice. c NK cells transfected with a Cy3-labeled miR-1249-3p mimic were co-cultured with 3T3-L1 adipocytes or AML12 cells in a Transwell TM plate (membrane pore = 0.4 mm) plate. Scale bar, 100 μm. d – g After transfecting with miR-1249-3p mimic, miR-NC, inhibitor, and inh-NC, the glucose uptake content of 3T3-L1 adipocytes ( d ) and glucose production content of AML12 cells ( f ) were measured, and the concentrations of IL-6, TNF-α, and IL-1β secreted by 3T3-L1 adipocytes ( e ) and AML12 cells ( g ) were detected by ELISA. Experiments were performed at least in triplicate, and the results are shown as the mean ± s.d. Student’s t -test was used to analyze the data. (* P < 0.05; ** P < 0.01; *** P < 0.001)

Article Snippet: Exosomal miRNA microarray analysis was performed at CapitalBio Technology, Inc. (Beijing, China) using Agilent Mouse miRNA 8 × 60K V21.0 Microarray (Agilent Technologies, California, USA).

Techniques: Derivative Assay, Microarray, Quantitative RT-PCR, Expressing, Transfection, Labeling, Cell Culture, Membrane, Enzyme-linked Immunosorbent Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Natural killer cell-derived exosomal miR-1249-3p attenuates insulin resistance and inflammation in mouse models of type 2 diabetes

doi: 10.1038/s41392-021-00805-y

Figure Lengend Snippet: Exosomal miR-1249-3p directly targets SKOR1 to mediate insulin sensitivity. a The potential targets of miR-1249-3p were predicted by integrating the results of two databases (TargetScan and miRDB). b Western blot analysis of SKOR1 in 3T3-L1 adipocytes and AML12 cells with the indicated treatments. c The wild-type and a mutated type of binding site between miR-1249-3p and SKOR1. d Relative luciferase activity of AML12 cells in the presence of indicated treatments. e Western blot analysis of SKOR1 expression in the VATs and livers of HFD mice after NCD-Exos or HFD-Exos treatment. f – i The effect of sh-SKOR1 on glucose uptake capacity in 3T3-L1 adipocytes ( f ), glucose production capacity in AML12 cells ( g ), and expression levels of IL-6, TNF-α, and IL-1β ( h , i ). j – q Glucose uptake content of 3T3-L1 adipocytes ( j ) and glucose production content of AML12 cells ( n ) with the indicated treatments were measured, as well as the expression levels of IL-6, TNF-α, and IL-1β ( k – m and o – q ). Experiments were performed at least in triplicate, and the results are shown as the mean ± s.d. Student’s t -test was used to analyze the data. (* P < 0.05; ** P < 0.01; *** P < 0.001)

Article Snippet: Exosomal miRNA microarray analysis was performed at CapitalBio Technology, Inc. (Beijing, China) using Agilent Mouse miRNA 8 × 60K V21.0 Microarray (Agilent Technologies, California, USA).

Techniques: Western Blot, Binding Assay, Luciferase, Activity Assay, Expressing

Journal: Signal Transduction and Targeted Therapy

Article Title: Natural killer cell-derived exosomal miR-1249-3p attenuates insulin resistance and inflammation in mouse models of type 2 diabetes

doi: 10.1038/s41392-021-00805-y

Figure Lengend Snippet: MiR-1249-3p relieves insulin resistance and inflammation via the SKOR1-SMAD6-TLR4-NF-κB axis. a – c After transfection with a miR-1249-3p mimic, miR-NC, or specific siRNA to SKOR1 or SMAD6, qRT-PCR analysis of miR-1249-3p ( a ), SKOR1 ( b ) and SMAD6 ( c ) expression in 3T3-L1 adipocytes cells with the indicated treatments was performed. d , g The expression of p-p65 and p65 in 3T3-L1 adipocytes cells with the indicated treatments was performed by western blot. e , f , h IL-1β, IL-6, and TNF-α expression in 3T3-L1 adipocytes cells subjected to the indicated treatments was assessed by ELISA. i The signaling pathway through which NK-derived exosomal miR-1249-3p regulates insulin resistance in type 2 diabetes mice. Experiments were performed at least in triplicate, and the results are shown as the mean ± s.d. Student’s t -test was used to analyze the data. (* P < 0.05; ** P < 0.01; *** P < 0.001)

Article Snippet: Exosomal miRNA microarray analysis was performed at CapitalBio Technology, Inc. (Beijing, China) using Agilent Mouse miRNA 8 × 60K V21.0 Microarray (Agilent Technologies, California, USA).

Techniques: Transfection, Quantitative RT-PCR, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay