Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Human Enterovirus in the Gastrocnemius of Patients With Peripheral Arterial Disease

doi: 10.1161/JAHA.113.000082

Figure Lengend Snippet: Viral RNA copy numbers were determined by RT‐qPCR. (A) Viral RNA copy numbers were compared among the Stage groups. (B) Single‐step growth curves of PAD‐HEV were examined at the various time points in the primary HSkm cells. Viral RNA copy numbers were determined using in vitro transcripts (see more detail in Supplemental Materials). PAD‐HEV1 (Stage III), PAD‐HEV2 (Stage II), PAD‐HEV3 and 4 (Stage IV). RT‐qPCR indicates reverse‐transcription real‐time quantitative polymerase chain reaction; PAD, peripheral arterial disease; HEV, human enterovirus; HSkm, primary human skeletal muscle; CVB1, coxsackievirus B1.

Article Snippet: HeLa cells (ATCC) were used for HEV propagation, HEV detection, and infectivity experiments., – Primary human skeletal muscle (HSkm) cells, cultured in MyoTonic Growth Medium (Cook MyoSite), also were used to study virus infectivity.

Techniques: Quantitative RT-PCR, In Vitro, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: International Journal of Medical Sciences

Article Title: LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2

doi: 10.7150/ijms.114095

Figure Lengend Snippet: Expression levels of cardiac markers and cardiac transcription factors in LAP2a CRISPR clones after 7 days in differentiation medium . ( A ) Whole cell protein extracts were prepared from CRISPR clones (WT +/+, LAP2a +/- and LAP2a -/-) grown in proliferation (Prolif.) or incubated for 7 days in differentiation medium (Diff.) and analysed by western blot. The latter were revealed either with mouse anti Actin alpha 1 cardiac muscle Ab and successively with mouse anti cardiac troponin T monoclonal Ab, or with mouse anti Myosin-2 Ab. Red Ponceau staining prior to incubation with antibodies is also shown. ( B ) The graphs depict the relative protein amount (mean ± s.e.m normalized to Red Ponceau) for a1 cardiac Actin, cardiac troponin T2 (TNNT2) and Myosin-2 in cells grown in proliferation (P) or differentiation (D) medium. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). The graphs present the individual values and means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 4 or 5 independent differentiation experiments for the LAP2a -/- clone). * p<0.05, ** p<0.01 (Mann Whitney test). ( C ) The graphs depict relative mRNA levels normalized to Hmbs for Actc1 , Tnnt2 and Myh7 in cells grown in proliferation (P) or differentiation (D) medium, as indicated. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). Each dot in the graphs represents the average value of a technical triplicate for RT-qPCR. The graphs also show the means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 4 or 5 independent differentiation experiments for the LAP2a -/- clone). ** p<0.01, *** p<0.001 (Mann Whitney test). ( D ) The graphs depict relative mRNA levels normalized to Hmbs for Gata4 , Mef2c and P300 in cells grown in proliferation (P) or differentiation (D) medium, as indicated. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). Each dot in the graphs represents the average value of a technical triplicate for RT-qPCR. The graphs also show the means ± s.e.m. (N = 3 independent differentiation experiments for each WT +/+ clone, N = 1 or 4 independent differentiation experiments for each LAP2a +/- clone; N = 4 independent differentiation experiments for the LAP2a -/- clone). * p<0.05, ** p<0.01, *** p<0.001 (Mann Whitney test).

Article Snippet: We used the following primary antibodies, according to the manufacturer's instructions: mouse anti-Actin alpha 1 cardiac muscle antibody (Ab) (33-32R, Novus Biological, BioTechne France), mouse anti-Histone H3 Ab (1G1, sc-517576; Santa Cruz Biotechnology, Germany), rabbit anti-Ki67 Ab (NB500-170; Novus Biological, BioTechne France), mouse anti-Lamin A/C Ab (4C11; Cell Signaling Technology, USA); rabbit anti-TMPO/LAP2 Ab (14651-1-AP; Proteintech Europe, United Kingdom) which recognizes all TMPO isoforms (a,b,g); mouse anti-Myosin-2 Ab (MF20, 14-6503-80, Life Technologies Europe, United Kingdom), rabbit anti-LAP2 alpha Ab (IQ175; Immuquest, Europe, United Kingdom); rabbit anti-Lamin B1 Ab ; rabbit anti-LEMD2 Ab (HPA017340, Merck Sigma, France), mouse anti-cardiac troponin T monoclonal Ab (13-11; MA512960 , Life Technologies, France) and phalloidin (P2141-Sigma).

Techniques: Expressing, CRISPR, Clone Assay, Incubation, Western Blot, Staining, MANN-WHITNEY, Quantitative RT-PCR

Journal: International Journal of Medical Sciences

Article Title: LAP2 Isoform Profile in Heart Ageing and in Cardiac Cell Proliferation and Differentiation: Input From CRISPR-Cas9-mediated LAP2a Knockdown in H9C2

doi: 10.7150/ijms.114095

Figure Lengend Snippet: Changes in LAP2a and LAP2b expression levels in vitro upon cardiac differentiation. ( A ) mRNAs were prepared from CRISPR clones (WT +/+, LAP2a +/- and LAP2a -/-) grown in proliferation (P) or incubated for 7 days in differentiation medium (D). Their relative amount was evaluated by RT-qPCR using specific primers (see Materials and Methods). The graphs depict relative mRNA amount (normalized to Hmbs ) for Lap2a and Lap2b. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). For each analysis, samples of LAP2a +/+ in proliferation (green circle) were used to arbitrarily define a reference value equal to 1.00. Each dot in the graphs represents the average value of a technical triplicate for RT-qPCR. The graphs also show the means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 3 or 4 independent differentiation experiments for the LAP2a -/- clone). * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001 (Mann Whitney test). ( B ) Whole cell protein extracts were prepared from CRISPR clones (WT +/+, LAP2a +/- and LAP2a -/-) grown in proliferation (Prolif., P) or incubated for 7 days in differentiation medium (Diff., D) and analysed by western blot. The latter were revealed with the rabbit anti TMPO Ab to detect LAP2a (alpha), LAP2b (beta) and a short LAP2b-like isoform (bsh). ( C-E ) The graphs depict the relative protein amount (i.e. mean ECL signal intensity (a.u) ± s.e.m normalized to Red ponceau) for LAP2a, LAP2 b, LAP2a:LAP2b, and LAP2bsh as indicated. To summarise the data, the clones have been grouped into 3 categories: WT +/+ (21B1, 22A11), LAP2a +/- (22G2, 22G3 and 21H4) and LAP2a -/- (22B3). For each analysis, samples of LAP2a +/+ in proliferation were used to arbitrarily define a reference value equal to 1.00 (green circle). The graphs present the individual values and means ± s.e.m. (N = 4 or 5 independent differentiation experiments for each WT +/+ clone, N = 2 to 4 independent differentiation experiments for each LAP2a +/- clone; N = 3 to 5 independent differentiation experiments for the LAP2a -/- clone). * p<0.05; ** p<0.01; *** p<0.001 (Mann Whitney test).

Article Snippet: We used the following primary antibodies, according to the manufacturer's instructions: mouse anti-Actin alpha 1 cardiac muscle antibody (Ab) (33-32R, Novus Biological, BioTechne France), mouse anti-Histone H3 Ab (1G1, sc-517576; Santa Cruz Biotechnology, Germany), rabbit anti-Ki67 Ab (NB500-170; Novus Biological, BioTechne France), mouse anti-Lamin A/C Ab (4C11; Cell Signaling Technology, USA); rabbit anti-TMPO/LAP2 Ab (14651-1-AP; Proteintech Europe, United Kingdom) which recognizes all TMPO isoforms (a,b,g); mouse anti-Myosin-2 Ab (MF20, 14-6503-80, Life Technologies Europe, United Kingdom), rabbit anti-LAP2 alpha Ab (IQ175; Immuquest, Europe, United Kingdom); rabbit anti-Lamin B1 Ab ; rabbit anti-LEMD2 Ab (HPA017340, Merck Sigma, France), mouse anti-cardiac troponin T monoclonal Ab (13-11; MA512960 , Life Technologies, France) and phalloidin (P2141-Sigma).

Techniques: Expressing, In Vitro, CRISPR, Clone Assay, Incubation, Quantitative RT-PCR, MANN-WHITNEY, Western Blot

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Haplodeficiency of Ataxia Telangiectasia Mutated Accelerates Heart Failure After Myocardial Infarction

doi: 10.1161/JAHA.117.006349

Figure Lengend Snippet: Senescence is involved in post–myocardial infarction ( MI) cardiac remodeling in mouse heart. A, Real‐time polymerase chain reaction (PCR) analysis of ataxia telangiectasia mutated (ATM), p53, and p21 mRNA levels at days 0, 3, and 7 after MI . B, Real‐time PCR analysis of ATM , p53, and p21 mRNA levels in sham mouse and different regions (remote area, border area, and infarct area) at day 7 post MI. n=4 in each group. * P <0.05 vs the day 0 or sham group. Western blot analysis and bar graph of (C) p21 expression in wild‐type mouse hearts at indicated times after sham or MI surgery, and (D) p21 expression in different regions of wild‐type mouse hearts 7 days after sham or MI surgery. E, Representative images and (F) statistical analysis of senescence‐associated β‐galactosidase (SA‐β‐gal) staining in mouse hearts at day 7 after sham or MI surgery. n=4 in each group. * P <0.05 vs the sham group, ** P <0.01 vs the sham group. G, Representative images of costaining of SA ‐β‐gal and α‐smooth muscle actin (α‐ SMA )/α‐actinin staining in heart section at day 7 after MI surgery. Bar=50 μm.

Article Snippet: Briefly, heart sections (5 μm) from paraffin‐embedded tissues were deparaffinized and incubated with anti‐mouse monoclonal antibodies against α‐smooth muscle actin (α‐SMA) (1:200 dilution, Santa Cruz Biotechnology) and CD31 (1:200 dilution, Cell Signaling Technology) at 4°C overnight.

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Staining

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Haplodeficiency of Ataxia Telangiectasia Mutated Accelerates Heart Failure After Myocardial Infarction

doi: 10.1161/JAHA.117.006349

Figure Lengend Snippet: Ataxia telangiectasia mutated (ATM) haplodeficiency increases myofibroblast accumulation and decreases fibroblast senescence both in vivo and in vitro. A, Quantitative real‐time polymerase chain reaction (PCR) was performed to detect the mRNA level of α‐smooth muscle actin (α‐ SMA ), collagen I, and collagen III in the hearts of ATM +/+ and ATM +/− mice at the indicated time points post‐myocardial infarction (MI). n=4 in each group. * P <0.05, # P <0.01 vs the corresponding sham group; † P <0.05 vs the control MI group. B, Western blot analysis of α‐ SMA levels in the hearts of ATM +/+ and ATM +/− mice at the indicated time points post MI . C, Representative collagen I–stained sections of infarct, border, and remote size 7 days after MI in ATM +/+ and ATM +/− mice. D, Western blot analysis of p21 levels in the hearts of ATM +/+ and ATM +/− mice at the indicated time points post MI . E, Representative images and statistical analysis of senescence‐associated β‐galactosidase (SA‐β‐gal) staining in cardiac sections from ATM +/+ and ATM +/− mice after MI . F, Representative images of costaining of p19 or p‐p53 with α‐ SMA in ATM +/+ and ATM +/− mice after MI . G, Representative images and statistical analysis of SA ‐β‐gal staining in isolated cardiac fibroblasts after hypoxia/oxygenation treatment and (H) cardiac fibroblasts from ATM +/+ and ATM +/− mice after MI surgery. n=4 in each group. * P <0.05 vs the corresponding ATM +/− group. Bar=50 μm.

Article Snippet: Briefly, heart sections (5 μm) from paraffin‐embedded tissues were deparaffinized and incubated with anti‐mouse monoclonal antibodies against α‐smooth muscle actin (α‐SMA) (1:200 dilution, Santa Cruz Biotechnology) and CD31 (1:200 dilution, Cell Signaling Technology) at 4°C overnight.

Techniques: In Vivo, In Vitro, Real-time Polymerase Chain Reaction, Control, Western Blot, Staining, Isolation

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Summary of glucose transporter family members

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques:

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Schematic of insulin-induced translocation of glucose transporter 4 from cytosol to the cell membrane. The binding of insulin to its receptors initiates a signal transduction cascade, which results in the activation of Akt. Akt acts on the glucose transporter 4 (GLUT4) containing vesicles in the cytosol to facilitate their fusion with the cell membrane. When more GLUT4 molecules are present in the membrane, the rate of glucose uptake is elevated. GLUT4: Glucose transporter 4.

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Translocation Assay, Binding Assay, Transduction, Activation Assay

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Recent studies of glucose transporter 4 expression and translocation in the skeletal muscle

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Expressing, Translocation Assay, Western Blot, Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Immunoprecipitation, Isolation, Transgenic Assay, Immunofluorescence, Microscopy, Electron Microscopy, Injection, In Vitro, Activation Assay, Labeling, Protease Inhibitor

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: The movement of glucose transporter 4 in adipocytes. Adipose tissue is made of adipocytes. In adipocytes, glucose transporter 4 (GLUT4) can be found in the cell membrane and in the cytosol. The translocation of GLUT4 from cytosolic vesicles to the cell membrane leads to elevated glucose uptake, whereas endocytosis brings GLUT4 back to the cytosol. ( 1): In unstimulated cells, GLUT4 containing membrane portions are internalized in an endocytosis manner to generate vesicles containing GLUT4. GLUT4 vesicles are internalized into early (or sorted) endosomes. They can enter the recovery endoplasmic body, and follow the retrograde pathway to the trans-Golgi network and endoplasmic reticulum-Golgi intermediate compartment or other donor membrane compartments. (2): GLUT4 vesicles derived from the donor membrane structures are secured by tether containing a UBX domain for GLUT4 (TUG) protein. (3): During insulin signal stimulation, GLUT4 vesicles are released and loaded onto the microtubule motor to be transferred to the plasma membrane. The continuous presence of insulin leads to the direct movement of these vesicles to the plasma membrane. (4): GLUT4 vesicles are tethered to motor protein kinesin and other proteins. A stable ternary SNARE complex forms when this occurs. (5): The stable ternary SNARE complex is docked on the target membrane. (6): The docked vesicles rely on SNARE to move to and fuse with the target membrane[ , , ]. GLUT4: Glucose transporter 4.

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Translocation Assay, Derivative Assay

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Recent studies of effects of bioactive compounds and chemical drugs on glucose transporter 4 expression and translocation in adipocytes

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Expressing, Translocation Assay, Fluorescence, Immunostaining, Western Blot, Inhibition, Immunoprecipitation, Real-time Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Immunofluorescence, In Vitro, In Vivo, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Activity Assay, Produced, Microscopy, Plasmid Preparation, Flow Cytometry

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Recent studies of mechanisms of glucose transporter 4 expression and translocation in adipocytes

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Expressing, Translocation Assay, Real-time Polymerase Chain Reaction, Electrophoretic Mobility Shift Assay, Immunohistochemistry, Western Blot, Transfection, Northern Blot, Nuclear Run-on Assay, Plasmid Preparation, Flow Cytometry, Fluorescence, Microscopy, Over Expression, Isolation, Concentration Assay, Immunofluorescence

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Recent studies of glucose transporter 4 expression and translocation in the heart

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Expressing, Translocation Assay, Positive Control, Inhibition, Activation Assay, Isolation, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, Immunohistochemistry

Journal: World Journal of Biological Chemistry

Article Title: Current understanding of glucose transporter 4 expression and functional mechanisms

doi: 10.4331/wjbc.v11.i3.76

Figure Lengend Snippet: Recent studies of glucose transporter 4 expression and translocation in the brain

Article Snippet: Western blot. , Whole cell and cell fractions from rat L6 and mouse C2C12 muscle cells, and soleus muscle of hind limb from mice. Anti-GLUT4 from Santa Cruz. Biotechnology (1:1000). , Whole cell lysate vs membrane fractions. Treatments without or with insulin or AICAR. , GLUT4 translocation occurs in L6 myotubes and 3T3-L1 adipocytes stimulated by insulin and AICAR. GLUT4 translocation occurs in muscle at 15 to 30 minutes and in adipose tissue at 15 minutes after glucose treatment. , [ ] .

Techniques: Expressing, Translocation Assay, Knock-Out, Western Blot, Cell Culture, Activity Assay, Immunocytochemistry, Real-time Polymerase Chain Reaction, Immunofluorescence, Immunohistochemistry, Positive Control, Negative Control, Microscopy, Transgenic Assay

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: Mexo-P regulates vascular calcification in a let-7b-5p-dependent way. ( A ) Volcano plots show the result of miRNA sequencing between Mexo- and Mexo-P-treated MOVAS. ( B ) qRT-PCR validation of potential target miRNAs between Mexo- and Mexo-P-treated HASMCs (unpaired t -test, n = 5). ( C ) Expression levels of let-7b-5p in the artery tissue of mildly, moderately, or severely calcified CKD patients (unpaired t -test, n > 3). ARS staining ( D ) and calcium contents (( E ), one-way ANOVA, n = 4) of isolated mouse thoracic aortas, cultured in a calcifying medium (3 mM Pi) for 7 days, pretreated with let-7b-5p agomir or antagomir for 6 h. Representative images of ARS staining ( F ) and ARS quantification ( G ) of MOVAS treated with let-7b-5p-mimic or control (mimic-nc) and cultured in the calcifying medium for 7 days (Mann–Whitney test, n = 4). Representative images of ARS staining ( H ) and ARS quantification ( I ) of MOVAS treated with let-7b-5p-inhibitor or control (inhibitor-nc) and cultured in the calcifying medium for 7 days (unpaired t -test, n = 4). Representative images of ARS staining ( J ) and ARS quantification ( K ) of HASMCs treated with let-7b-5p-mimic or control and cultured in the calcifying medium for 7 days (unpaired t -test, n = 4). Representative images of ARS staining ( L ) and ARS quantification ( M ) of MOVAS treated with let-7b-5p-inhibitor or control and cultured in the calcifying medium for 7 days (unpaired t -test, n = 4). Calcium content levels of calcifying-medium-cultured VSMCs (MOVAS, ( N ), HASMC, ( O )), stimulated with Mexo or Mexo-P in the presence or absence of let-7b-5p-mimic (one-way ANOVA, n = 4). * p < 0.05, *** p < 0.001, **** p < 0.0001, n.s. not significant. Scale bar, 40 μM.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Sequencing, Quantitative RT-PCR, Biomarker Discovery, Expressing, Staining, Isolation, Cell Culture, Control, MANN-WHITNEY

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: Let-7b-5p mitigates vascular calcification by regulating TGFBR1. ( A ) Volcano plots showing differentially regulated genes (DEGs) in RNA-seq of Mexo- and Mexo-P-treated MOVAS. ( B ) Venn diagram showing the intersection of miRBD-predicted potential let-7b-5p targets and DEGs. ( C ) mRNA levels of 10 potential target genes were detected by qRT-PCR in let-7b-5p-mimic- or mimic-nc-treated HASMCs (unpaired t -test, n > 3). ( D ) Transcription levels of four significantly regulated genes in ( C ) were detected in Mexo- or Mexo-P-treated HASMC (unpaired t -test, n > 3). ( E ) Representative images of ARS staining (bottom) and ARS quantification (top) of HASMCs, in which TGFBR1 and let-7b-5p levels were manipulated with TGFBR1 overexpression plasmid (TGFBR1-OE) or let-7b-5p-mimic treatment in calcifying medium (one-way ANOVA, n = 4). ( F ) Representative images of ARS staining (bottom) and ARS quantification (top) of HASMCs, in which TGFBR1 and let-7b-5p levels were manipulated with TGFBR1-si or let-7b-5p-mimic treatment in calcifying medium (one-way ANOVA, n = 4). Calcium content of HASMCs with altered TGFBR1 levels with TGFBR1-OE ( G ) or TGFBR1-si ( H ), with or without the treatment of let-7b-5p-mimic or let-7b-5p-inhibitor in calcifying medium (one-way ANOVA, n = 4). ( I ) Tergetscan database predicted binding sequences of let-7b-5p on TGFBR1-3′UTR. ( J ) Wildtype (wt) and two mutations (mut1 and mut2) of TGFBR1-3′UTR were inserted into luciferase vectors. Luciferase activity assay of HASMCs co-transfected with vectors carrying wt or mutated TGFBR1-3′UTR (mut1, ( K ), mut2, ( L )), renilla vectors, mimic-nc, or let-7b-5p-mimic for 6 h, medium change, and cultured for another 48 h before assay (two-way ANOVA, n > 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: RNA Sequencing, Quantitative RT-PCR, Staining, Over Expression, Plasmid Preparation, Binding Assay, Luciferase, Activity Assay, Transfection, Cell Culture

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: Manipulating TGFBR1 levels regulates vascular calcification in CKD. Transcription levels of RUNX2, ALPL, and SOX9 in MOVAS ( A ) and HASMCs ( C ) stimulated with 10 ng/mL TGFβ1 for 24 h after being transfected with TGFBR1 siRNA for 6 h (one-way ANOVA, n > 3). mRNA levels of RUNX2, ALPL, and SOX9 in MOVAS ( B ) and HASMCs ( D ) stimulated with 10 ng/mL TGFβ1 for 24 h after being transfected with TGFBR1 overexpression plasmid (TGFBR1-OE) for 48 h (one-way ANOVA, n > 3). Representative WB images ( E ) and semi-quantification ( F ) showing RUNX2 levels in aortas from CKD mice injected with either Tgfbr1-adv or control (GFP-adv, unpaired t -test, n = 3). Representative images of ARS staining ( G ) and calcium content ( H ) of aortas from CKD mice injected with either Tgfbr1-adv or control (unpaired t -test, n > 3). ( I ) HASMCs were treated with SB525334 (1 μM), RepSOX (25 μM), and SB431542 (1 μM) for 6 h, after being transfected with SMAD3-luciferase plasmids for 24 h, and luciferase activity was detected (unpaired t -test, n = 3). ( J ) SMAD3-luciferase plasmid-transfected HASMCs were stimulated with 10 ng/mL TGFβ1 and different doses of SB525334 for 6 h, and luciferase activity was detected (one-way ANOVA, n > 3). ( K ) HASMCs were stimulated with 10 ng/mL TGFβ1 plus 1 μM SB525334 for 0, 3, 6, 12, 24, or 48 h, and RUNX2 mRNA levels were detected by qRT-PCR (one-way ANOVA, n = 5). In vitro calcification model showing the SB525334 (1 μM) inhibition of 10 ng/mL TGFβ1-induced HASMC calcification (( L ), ARS staining, ( M ), calcium content, one-way ANOVA, n = 6). Mice were orally administrated with 30 mg/kg SB525334 (n = 5) or vehicle (n = 5) daily; simultaneously with CKD modeling, aortas were collected for ARS staining ( N ), calcium content assay (( O ), unpaired t -test, n > 3), and Runx2, Alpl, and Sox9 mRNA levels detection (( P ), unpaired t -test, n > 3). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant. Scale bar, 40 μM.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Transfection, Over Expression, Plasmid Preparation, Injection, Control, Staining, Luciferase, Activity Assay, Quantitative RT-PCR, In Vitro, Inhibition

Journal: Cells

Article Title: High-Phosphate-Stimulated Macrophage-Derived Exosomes Promote Vascular Calcification via let-7b-5p/TGFBR1 Axis in Chronic Kidney Disease

doi: 10.3390/cells12010161

Figure Lengend Snippet: TGFBR1 promotes vascular calcification through the SMAD3/RUNX2 pathway. qRT-PCR results showing that SIS-3 reversed TGFβ1-induced upregulation of RUNX2 levels in HASMCs ( A ) and MOVAS (( B ), one-way ANOVA, n = 5). Western blot images showing that different doses of SMAD3 inhibitor SIS-3 alleviated TGFβ1-induced RUNX2 level increase and SMAD2/3 phosphorylation in HAMSCs ( C ) and MOVAS ( D ). ( E ) The JASPAR database predicted the binding site of SMAD3 on RUNX2 promoter, and the mutation was generated. ( F ) HASMCs were transfected with Renilla plasmid, wildtype RUNX2-promoter luciferase plasmid, and different doses of SMAD3-OE plasmid for 24 h, and luciferase activity was detected (one-way ANOVA, n = 5). ( G ) HASMC was transfected with Renilla plasmid, SMAD3-OE plasmid (3 ng per well of 12-well plate), and wildtype (wt) or mutated (mut) RUNX2-promoter luciferase plasmid, and luciferase activity was detected (Mann–Whitney test, n = 6). ** p < 0.01, *** p < 0.001, **** p < 0.0001, n.s. not significant.

Article Snippet: The mouse vascular smooth muscle (MOVAS) cell line (CRL-2797TM) and human aortic smooth muscle cell (HASMC) strain (PCS-100-012TM) were from American Type Culture Collection (ATCC, Rockville, MD, USA).

Techniques: Quantitative RT-PCR, Western Blot, Phospho-proteomics, Binding Assay, Mutagenesis, Generated, Transfection, Plasmid Preparation, Luciferase, Activity Assay, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are localized and secreted by human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A Human lung tissue from control subjects, idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF was immune-stained with IL-11, IL-11Rα and αSMA and with secondary fluorescence antibodies. Representative images are showed. White colour represents co-localization of both antibodies. Yellow arrows indicate endothelial cells. B HPAECs and C HPASMCs were isolated from pulmonary arteries of control subjects, IPF and PH associated to IPF patients and cultured until passage 1. Cell culture supernatants were collected to measure IL-11 by ELISA. Data are presented as scatter dot blot with median and interquartile range values of n = 6 patients in each group. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Staining, Fluorescence, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and IL-11Rα are increased in whole lung homogenates, isolated pulmonary arteries and serum of patients with idiopathic pulmonary fibrosis (IPF) and pulmonary hypertension (PH) associated to IPF. The protein expression of IL-11 and IL-11Rα in A , B isolated pulmonary arteries (70–500 µm of internal diameter), C , D serum, and E , F lung tissue homogenates. Protein expression was measured using ELISA kits. H CD31 protein expression was measured in isolated pulmonary arteries as endothelial cells marker by ELISA. H , I Human lung tissue from control subjects, IPF and PH associated to IPF was immune-stained with IL-11, IL-11Rα and alpha smooth muscle actin (αSMA) antibodies. Representative images are showed from non-fibrotic lung areas and fibrotic areas. J Vascular wall thickening was quantified in a total of 20–30 pulmonary arteries per patient. K , L Immunohistochemical score quantification of IL-11 and IL-11Rα in a total of 20–30 pulmonary arteries per patient. Scale bar: 100 µm. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison. M Spearman ρ correlation of IL-11 expression in isolated pulmonary arteries from PH + IPF and mean pulmonary artery pressure (mPAP). N indicates the number of patients in each graph

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Marker, Staining, Immunohistochemical staining, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: SiRNA-IL-11 transiently transfection attenuates bleomycin-induced lung fibrosis and pulmonary hypertension in transgenic Tie2-GFP mice. Wild-type (WT) siRNA(−) Tie2-GFP mice and IL-11-KO siRNA-IL-11 Tie2-GFP mice received a single intratracheal dose of bleomycin (1.5 U/kg) on day 1 ( n = 11) during 14 days. siRNA-IL-11 was administered intravenously and intranasally three times a week from day 1 to day 14. At day 14 the following parameters were measured. A Masson’s trichrome histological images are showed. Scale bar: 100 µm. B Ashcroft score lung fibrotic index, C hydroxyproline amount in lung tissue D right ventricular systolic pressure (RVSP) mmHg, E right ventricular (RV) hypertrophy measured by the ratio of RV/left ventricular (LV) + septo in mg/mg, F pulmonary artery remodeling and G inflammatory cells in bronchoalveolar lavage fluid (BALF) were measured. H Immunohistochemical analysis of αSMA, IL-11 and IL-11Rα. Scale bar: 50 µm. Black arrows show pulmonary arteries. I Co-immunofluorescence of αSMA/Tie2-GFP. Scale bar: 25 µm. White arrows indicates co-localizations. Data are presented as scatter dot blot with median and interquartile range values. P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Transfection, Transgenic Assay, Immunohistochemical staining, Immunofluorescence, Dot Blot

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: IL-11 and soluble IL-11Rα induce human pulmonary artery endothelial cell (HPAEC) to mesenchymal transition (EnMT) and human pulmonary artery smooth muscle cell (HPASMC) to myofibroblast-like transition. A HPAEC and B HPASMC were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or their combination during 48 h replacing culture medium and stimulus each 24 h. Experiments were done between passages 2–3. Gene mRNA transcripts of different genes measured by quantitative PCR (qPCR) as 2 −ΔCt . Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin for protein. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 4 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα activates intracellular signal. A Human pulmonary artery endothelial cells (HPAEC) and B human pulmonary artery smooth muscle cells (HPASMC) were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination during 30 min. Experiments were done between passages 2–3. Protein expression levels were analysed by western blotting. Data are shown as the ratio compared to β-actin or non-phosphorylated protein as indicate. Representative blots are sowed. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3 control subjects performed in triplicate). P -values are based on the Mann Whitney test (two groups) or the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Expressing, Western Blot, Dot Blot, MANN-WHITNEY

Journal: Respiratory Research

Article Title: IL-11 system participates in pulmonary artery remodeling and hypertension in pulmonary fibrosis

doi: 10.1186/s12931-022-02241-0

Figure Lengend Snippet: rhIL-11 and soluble rhIL-11Rα promotes time-dependent proliferation and senescence in human pulmonary artery endothelial cells (HPAEC) and smooth muscle cells (HPASMC). A HPAECs or B HPASMCs were isolated from control donor subjects and stimulated with rhIL-11 5 ng/ml, rhIL-11Rα 10 ng/ml or its combination at indicated times. Experiments were done between passages 2–3. Cell proliferation was measured by the BrDU kit at 24 h, 48 h, 72 h and 96 h. Cell senescence was measured after 72 h of cell stimulation using β-galactosidase histology and P21 expression. Results were expressed as % senescence (β-galactosidase blue positive cells) relative to the total number of cells in each field. P21 expression was measured by quantitative PCR (qPCR) as 2 −ΔCt and western blot. Data are presented as scatter dot blot with median and interquartile range values (for primary cells, n = 3–4 control subjects performed in triplicate). P -values are based on the Kruskal–Wallis test and Dunn’s post-hoc test for multiple comparison

Article Snippet: For in vitro studies, HPAECs, HPASMCs and mice lung fibroblasts were stimulated with recombinant human IL-11 (rhIL-11, 5 ng/ml; cat. n. SRP3072, Sigma Aldrich), recombinant mice IL-11 (rmIL-11, 5 ng/ml; cat. n. Z03052-1, GeneScript), recombinant human IL-11RΑ (rhIL-11RΑ 10 ng/ml; cat. n. H00003590-P01, NOVUSBIO), recombinant mouse IL-11RΑ (rmIL-11RΑ 10 ng/ml; cat. n. 50,075-M08H, SinoBiological), or combinations for the indicated times, replacing culture medium and stimulus every 24 h. Selected concentrations were from concentration dependent curves on airway epithelial cells (data not shown) and literature [ ].

Techniques: Isolation, Cell Stimulation, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Dot Blot

Journal:

Article Title: Acute Humanin Therapy Attenuates Myocardial Ischemia and Reperfusion Injury in Mice

doi: 10.1161/ATVBAHA.110.205997

Figure Lengend Snippet: HNG improves cardiomyocyte survival in vitro and decreases apoptosis in response to Daunorubicin. Effect of HNG on A) Cardiomyocyte survival as assessed by cell viability assay, * p < 0.05 Dauno compared to Dauno+HNG, and B) early markers of apoptosis as assessed by CaspASE FITC -VAD-FMK in situ apoptosis assay.

Article Snippet: Effect of HNG on cardiomyocyte survival and apoptosis in vitro Rat heart myocardium cell line H9c2 (2-1) (cat# CRL-1446) was purchased from ATCC.

Techniques: In Vitro, Viability Assay, In Situ, Apoptosis Assay