mechanics mouse cardiomyocytes Search Results


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  • 99
    Thermo Fisher dmem
    Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 147955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore fatty acid free bsa
    Adiponectin promotes cardiomyocyte and Heart ATTAC survival ( a ) Female heart ATTAC transgenic mice crossed into indicated adiponectin backgrounds were challenged with AP20187 (0.010 μg/kg, IP) and survival was recorded as a Kaplan-Meier plot (n=12/group). ( b ) Ceramide was quantified from left ventricle or serum and normalized to the average content from adiponectin wildtype mice (63.9 pmol/mg in left ventricle, 9.5 pmol/μL in serum) (n=12/group). ( c ) Sphingosine, dihyrosphingosine, S1P, and dihydroS1P were quantified in left ventricle of WT (+/+), adiponectin (−/+), and adiponectin null mice (n=6/group). ( d ) Male HEART-ATTAC mice were treated with <t>myriocin</t> (0.3 mg/kg, IP), FTY720 (1 mg/kg, IP), S1P (1mg/kg, IP) or PBS immediately prior to injection with AP20187 (0.05 μg/kg, IP) and survival was recorded as a Kaplan-Meier plot (n=10/group). Additionally, treating HEART-ATTAC mice with S1P (1mg/kg, IP) just prior to AP20187 treatment prevented death in 100% of the animals tested (n=15) ( e ) Primary cardiomyocytes were isolated from heart ATTAC transgenic pups. After 72 hours of maintenance, cells were washed and treated with 2% <t>BSA</t> conjugated with: C2-ceramide (10 μM), myriocin (10 μM), palmitate (375 μM), or palmitate plus myriocin. PBS, adiponectin (3 μg/mL), or S1P (1 μM) were immediately added. Apoptosis was initiated by the addition of AP20187 (6.25 ng/mL), and viability was determined after 16 hours (n=6/group from 3 separate experiments). * denotes significant (p
    Fatty Acid Free Bsa, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3848 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IonOptix cardiomyocytes
    Effect of TGF-β on <t>cardiomyocyte</t> contractile function in myocytes isolated from FVB and metallothionein (MT) mice (maintained at normal temperature). A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal of relengthening (− dL/dt); E: Time-to-PS (TPS) and F: Time-to-90% relengthening (TR 90 ). Mean ± SEM, n = 73 – 80 cells from 3 mice per group, * p
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    Thermo Fisher collagenase type ii
    Effect of TGF-β on <t>cardiomyocyte</t> contractile function in myocytes isolated from FVB and metallothionein (MT) mice (maintained at normal temperature). A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal of relengthening (− dL/dt); E: Time-to-PS (TPS) and F: Time-to-90% relengthening (TR 90 ). Mean ± SEM, n = 73 – 80 cells from 3 mice per group, * p
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    Thermo Fisher collagenase
    Effect of TGF-β on <t>cardiomyocyte</t> contractile function in myocytes isolated from FVB and metallothionein (MT) mice (maintained at normal temperature). A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal of relengthening (− dL/dt); E: Time-to-PS (TPS) and F: Time-to-90% relengthening (TR 90 ). Mean ± SEM, n = 73 – 80 cells from 3 mice per group, * p
    Collagenase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3819 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher balanced salt solution
    Effect of TGF-β on <t>cardiomyocyte</t> contractile function in myocytes isolated from FVB and metallothionein (MT) mice (maintained at normal temperature). A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal of relengthening (− dL/dt); E: Time-to-PS (TPS) and F: Time-to-90% relengthening (TR 90 ). Mean ± SEM, n = 73 – 80 cells from 3 mice per group, * p
    Balanced Salt Solution, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7705 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher collagenase iv
    Effect of TGF-β on <t>cardiomyocyte</t> contractile function in myocytes isolated from FVB and metallothionein (MT) mice (maintained at normal temperature). A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal of relengthening (− dL/dt); E: Time-to-PS (TPS) and F: Time-to-90% relengthening (TR 90 ). Mean ± SEM, n = 73 – 80 cells from 3 mice per group, * p
    Collagenase Iv, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4955 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore 2 3 butanedione monoxime
    Rab3D release and actin coating of zymogen granules are interdependent events. ( a – c ) Double localization of rab3D and filamentous actin in tissue challenged with carbamylcholine (10 μM, 1 h). A close-up of part of an acinar lumen (asterisk) and the underlying zymogen granule field is shown. Rab3D immunofluorescence is depicted in a , and the corresponding phalloidin fluorescence is shown in b . The merge of the rab3D and phalloidin fluorescence images is shown in c . Note that the actin-coated granule (arrow) is not outlined by rab3D immunoreactivity, in contrast to the other zymogen granules. ( d ) Rab3D immunofluorescence in an acinus of tissue that was stimulated with carbamylcholine, showing absence of rab3D immunoreactivity on the apical membrane surrounding the lumen (asterisk). ( e ) Rab3D immunofluorescence in an acinus stimulated with carbamylcholine in the presence of cytochalasin D. The dramatically enlarged lumen (asterisks) is now outlined by strong rab3D staining. ( f – h ) Double localization of rab3D and filamentous actin in an acinus stimulated with carbamylcholine in the presence of latrunculin B and <t>2,3-butanedione</t> monoxime. Phalloidin fluorescence is shown in ( f ). Note the presence of numerous actin-coated granules packed in clusters. Also note that the acinar lumen has not expanded. The corresponding rab3D immunofluorescence is depicted in g , and the merge between both fluorescence images is shown in h . Boxes in f and g outline an area that is shown at higher magnification in i (phalloidin), j (rab3D), and k (merge). Note that the actin-coated granules (two are indicated by arrows) are rab3D-immunoreactive. [Bars = 2 μm ( b , d , and i ) and 5 μm ( e and f ).]
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    Thermo Fisher fkbp12 6
    Overexpression of <t>FKBP12.6</t> significantly protects cardiomyocytes from AngII‐induced hypertrophy through inhibiting the Ca 2+ /CaMKII, AKT/GSK3β and AKT/mTOR signalling pathways in H9c2 cells. FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with PBS or with 200 nmol/L AngII for 20 min. The expressions of the total and phosphorylated CaMKII (A), AKT, GSK3β and mTOR (D) were detected by Western blot. The quantitative expressions of total and phosphoryated CaMKII, AKT and GSK3β protein were performed by density analysis, and the protein expression of p‐CaMKII (B), p‐AKT (C), p‐GSK3β (E) and p‐mTOR (F) was normalized to total CaMKII, AKT, GSK3β and mTOR. GAPDH was used as the loading controls. The data represent the mean ± SEM from three independent experiments, * P
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    cxcr4  (Abcam)
    94
    Abcam cxcr4
    <t>AAV9.CXCR4</t> dose response in vivo . AAV9.CXCR4 WT was injected via the tail vein with increasing viral genomes/mouse, ranging from 2e9 to 3e11. Hearts were harvested four weeks post-injection. (A) CXCR4 mRNA was quantified by RT-PCR. (B) CXCR4 protein expression was assessed by western blot. (C) Immunostaining of CXCR4 in the heart indicated proper membrane localization.
    Cxcr4, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 3526 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    IonOptix ionoptix soft edge system
    <t>AAV9.CXCR4</t> dose response in vivo . AAV9.CXCR4 WT was injected via the tail vein with increasing viral genomes/mouse, ranging from 2e9 to 3e11. Hearts were harvested four weeks post-injection. (A) CXCR4 mRNA was quantified by RT-PCR. (B) CXCR4 protein expression was assessed by western blot. (C) Immunostaining of CXCR4 in the heart indicated proper membrane localization.
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    Santa Cruz Biotechnology sirt1
    (A) Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L) and NAM (5 and 10 mmol/L). THP-1 cells were treated with LPS (10 μg/mL) for 4, 8, 12 h. <t>SIRT1</t> activity was determined by colorimetric assay. (*Represents P ≤ 0.05 LPS exposed cells in the absence or presence of DIQ; # represents P ≤ 0.05 LPS exposed cells treated with DIQ or DIQ and NAM.) Assay shown is representative of three experiments with similar results. (B) Representative autoradiograph of Western blot analysis for nuclear SIRT1 concentration in THP-1 cells treated with LPS in the presence or absence of DIQ (100 and 300 μmol/L). THP-1 cells were treated with LPS (10 μg/mL) for 4 and 8 h. The gel is representative of three experiments with similar results. (C) Nuclear proteins were extracted from WT and PARP1 −/− cells and SIRT1 activity was determined by colorimetric assay. (*Represents P ≤ 0.05 versus WT cells.) (D) Representative autoradiograph of Western blot analysis for supernatant HMGB1 levels in THP-1 cells treated with LPS in the presence or absence of SRT1720 (0.01 and 0.1 μmol/L), a SIRT1 activator. THP-1 cells were treated with LPS (10 μg/mL) for 18 h. The gel is representative of three experiments with similar results.
    Sirt1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 3877 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Epitomics foxo1
    Autophagy inhibition attenuates <t>FoxO1-induced</t> reduction in cell size and relative protein content. Cultured cardiomyocytes were transduced with Ad-FoxO1-WT or Ad-LacZ for 24 hours or Ad-sh-Beclin1 for 96 hours, and treated with 10 mM 3-methyladenine (3-MA) for 24 hours. A) Representative immunoblots. B) Densitometric analyses. C) Representative images of cardiomyocytes viewed under a light microscope. D) Relative cardiomyocyte cross-sectional area. E) Relative protein content. Data represent means from at least 6 different myocyte cultures. * p
    Foxo1, supplied by Epitomics, used in various techniques. Bioz Stars score: 92/100, based on 108 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology sglt1
    Cardiac <t>SGLT1</t> expression was regulated by AMPK. (A) Levels of Thr 172 phosphorylated AMPK a subunit (P-AMPKα), which reflect AMPK activity, were increased in 8 week old male WT FVB mice after administration of the AMPK activator AICAR (500 μg/kg IP, twice at a 3 h interval). (B) Concurrently, cardiac SGLT1 mRNA expression was increased in these mice after administration of AICAR ( n = 6 / group). (C) Levels of Thr 172 phosphorylated AMPK α subunit (P-AMPKα) were normalized in TG T400N /TG α2DN relative to TG T400N hearts at age 2 weeks. (D) Double transgenic mice (TG T400N /TG α2DN ) exhibited attenuation of cardiac glucose uptake ( n = 3 / group). (E) Increased SGLT1 mRNA expression in TG T00N hearts was attenuated in TG T400N /TG α2DN hearts, as assessed by QPCR ( n = 3 / group). (F) An immunoblot showed that the increased SGLT1 protein expression observed in TG T00N hearts was attenuated in TG T400N /TG α2DN hearts. (G) Densitometry analysis of the immunoblot shown in panel F. Chromatin immunoprecipitation (ChIP) showed that increased expression of SGLT1 in TG T00N hearts relative to WT was associated with increased binding of (H) HNF-1 and (I) Sp1 to the promoter of the SLC5A1 gene encoding SGLT1 ( n = 3 / group). Molecular weights (MW) on immunoblots were estimated by protein marker sizes. Coomassie blue staining was used to document the relative quantity of protein loaded for the immunoblots. *, P
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    alk1  (Abcam)
    91
    Abcam alk1
    Reduced <t>ALK1</t> Expression Does Not Affect Cardiac Hypertrophy or Myocardial Capillary Density A and B, Representative histological staining and quantification of LV cardiomyocyte cross sectional area in WT and Alk1 +/− mice after two weeks of TAC (n=6/group). C to E, LV mRNA levels of beta-myosin heavy chain (MHC), sarcoplasmic endoplasmic reticulum ATPase (SERCA) and calcineurin. F and G, Representative immunostaining and quantification of myocardial capillary density. p
    Alk1, supplied by Abcam, used in various techniques. Bioz Stars score: 91/100, based on 84 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology bcl 2
    O -GlcNAc modification is essential for cellular survival and <t>Bcl-2</t> translocation. A) Representative immunoblotting CTD110.6 and OGT and mean intensity of all O -GlcNAc proteins and OGT of the total whole cell lysate determined by densitometric analysis.
    Bcl 2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 14600 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher endothelin 1 et 1
    (a) <t>Endothelin-1</t> <t>(ET-1)</t> gene expression in MPS bioreactor constructs vs. constructs engineered in plate. (b) and (c) representative IHC pictures of PCL and PCL/HA based constructs respectively, stained with anti-pAKT antibody: positive red signals are pointed by arrows. Scale bar = 100 μm
    Endothelin 1 Et 1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Exosome Diagnostics hsp20
    Altered exosomal contents by overexpression of <t>Hsp20</t> in cardiomyocytes could be delivered to endothelial cells and native cardiomyocytes. Representative Western blots ( A ) and their quantitative results ( B ) showed that higher levels of Hsp20, survivin, and p-Akt encased in TG-Exo than WT-Exo. C : SOD1 levels were increased in TG-Exo, compared with WT-Exo ( n = 4 for A – C ). * P
    Hsp20, supplied by Exosome Diagnostics, used in various techniques. Bioz Stars score: 91/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Santa Cruz Biotechnology arrb2
    <t>Arrb2/miR-155/GSK3β</t> pathway is important in CSC-mediated cardiac repair. Isolated 2 × 10 5 Sca-1+ cells from WT or Arrb2-KO mice were injected immediately into infarcted and border zones of the mouse heart after myocardial infarction (MI). Hearts were then reperfused for 1 hr. After 2 weeks, 2-mm sections of hearts near the mid-ventricles were collected. ( A ) Fluoresence microscopy of hearts for WT mice with MI injected with WT Sca-1+ cells and stained with cTnT. Red shows cardiomyocytes; green shows injected Sca-1+ cells; blue shows DAPI-stained cell nuclei; scale bar, 40 μm; n = 6. ( B ) Western blot analysis of the expression of Arrb2, total Akt and p-Akt, and total and p-GSK3β. GADPH was a loading control. The column shows the quantification of the protein expression. Protein levels were normalized to GAPDH or total protein; n = 3; * P
    Arrb2, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher fetal bovine serum
    <t>Arrb2/miR-155/GSK3β</t> pathway is important in CSC-mediated cardiac repair. Isolated 2 × 10 5 Sca-1+ cells from WT or Arrb2-KO mice were injected immediately into infarcted and border zones of the mouse heart after myocardial infarction (MI). Hearts were then reperfused for 1 hr. After 2 weeks, 2-mm sections of hearts near the mid-ventricles were collected. ( A ) Fluoresence microscopy of hearts for WT mice with MI injected with WT Sca-1+ cells and stained with cTnT. Red shows cardiomyocytes; green shows injected Sca-1+ cells; blue shows DAPI-stained cell nuclei; scale bar, 40 μm; n = 6. ( B ) Western blot analysis of the expression of Arrb2, total Akt and p-Akt, and total and p-GSK3β. GADPH was a loading control. The column shows the quantification of the protein expression. Protein levels were normalized to GAPDH or total protein; n = 3; * P
    Fetal Bovine Serum, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 225914 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore monodansylcadaverine
    Confocal photomicrographs demonstrating internalized PNP (120 nm amidine-modified) and clathrin heavy chain in MDCK-II after 1 h of apical exposure to 176 μg/mL PNP in the absence of <t>dansylcadaverine</t> (Panel A). PNP appear in green, nuclei in blue and clathrin heavy chain in red. PNP colocalizes with clathrin heavy chain, appearing yellowish-green. Panel B illustrates the colocalization profile of PNP (green) and clathrin heavy chain (red), where color intensities from green and red channels are estimated along the line segment shown in panel A. As can be appreciated, green intensity peaks coincide with red intensity peaks. The numbers 1–5 in panel B correspond to those in panel A, respectively. Panel C represents a typical confocal image obtained from MDCK-II co-incubated apically with 200 μM dansylcadaverine and 120 nm amidine-modified PNP for 1 h. Comparatively few PNP (green) can be seen in Panel C. No colocalization of PNP and clathrin heavy chain (red) is detectable in Panel C.
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    Image Search Results


    Adiponectin promotes cardiomyocyte and Heart ATTAC survival ( a ) Female heart ATTAC transgenic mice crossed into indicated adiponectin backgrounds were challenged with AP20187 (0.010 μg/kg, IP) and survival was recorded as a Kaplan-Meier plot (n=12/group). ( b ) Ceramide was quantified from left ventricle or serum and normalized to the average content from adiponectin wildtype mice (63.9 pmol/mg in left ventricle, 9.5 pmol/μL in serum) (n=12/group). ( c ) Sphingosine, dihyrosphingosine, S1P, and dihydroS1P were quantified in left ventricle of WT (+/+), adiponectin (−/+), and adiponectin null mice (n=6/group). ( d ) Male HEART-ATTAC mice were treated with myriocin (0.3 mg/kg, IP), FTY720 (1 mg/kg, IP), S1P (1mg/kg, IP) or PBS immediately prior to injection with AP20187 (0.05 μg/kg, IP) and survival was recorded as a Kaplan-Meier plot (n=10/group). Additionally, treating HEART-ATTAC mice with S1P (1mg/kg, IP) just prior to AP20187 treatment prevented death in 100% of the animals tested (n=15) ( e ) Primary cardiomyocytes were isolated from heart ATTAC transgenic pups. After 72 hours of maintenance, cells were washed and treated with 2% BSA conjugated with: C2-ceramide (10 μM), myriocin (10 μM), palmitate (375 μM), or palmitate plus myriocin. PBS, adiponectin (3 μg/mL), or S1P (1 μM) were immediately added. Apoptosis was initiated by the addition of AP20187 (6.25 ng/mL), and viability was determined after 16 hours (n=6/group from 3 separate experiments). * denotes significant (p

    Journal: Nature medicine

    Article Title: The Pleiotropic Actions of Adiponectin are Initiated via Receptor-Mediated Activation of Ceramidase Activity

    doi: 10.1038/nm.2277

    Figure Lengend Snippet: Adiponectin promotes cardiomyocyte and Heart ATTAC survival ( a ) Female heart ATTAC transgenic mice crossed into indicated adiponectin backgrounds were challenged with AP20187 (0.010 μg/kg, IP) and survival was recorded as a Kaplan-Meier plot (n=12/group). ( b ) Ceramide was quantified from left ventricle or serum and normalized to the average content from adiponectin wildtype mice (63.9 pmol/mg in left ventricle, 9.5 pmol/μL in serum) (n=12/group). ( c ) Sphingosine, dihyrosphingosine, S1P, and dihydroS1P were quantified in left ventricle of WT (+/+), adiponectin (−/+), and adiponectin null mice (n=6/group). ( d ) Male HEART-ATTAC mice were treated with myriocin (0.3 mg/kg, IP), FTY720 (1 mg/kg, IP), S1P (1mg/kg, IP) or PBS immediately prior to injection with AP20187 (0.05 μg/kg, IP) and survival was recorded as a Kaplan-Meier plot (n=10/group). Additionally, treating HEART-ATTAC mice with S1P (1mg/kg, IP) just prior to AP20187 treatment prevented death in 100% of the animals tested (n=15) ( e ) Primary cardiomyocytes were isolated from heart ATTAC transgenic pups. After 72 hours of maintenance, cells were washed and treated with 2% BSA conjugated with: C2-ceramide (10 μM), myriocin (10 μM), palmitate (375 μM), or palmitate plus myriocin. PBS, adiponectin (3 μg/mL), or S1P (1 μM) were immediately added. Apoptosis was initiated by the addition of AP20187 (6.25 ng/mL), and viability was determined after 16 hours (n=6/group from 3 separate experiments). * denotes significant (p

    Article Snippet: Myriocin, fatty acid free BSA, palmitic acid, propidium iodide and Annexin VFITC were from Sigma.

    Techniques: Transgenic Assay, Mouse Assay, Injection, Isolation

    Adiponectin alters sensitivity to ceramide-induced apoptosis in INS-1 β-cells ( a ) INS-1 cells were washed and removed to 2% BSA, Palmitate (750 μM in 2% BSA), or C2-Ceramide (50 μM in 2% BSA). Adiponectin (3 μg/mL) or PBS was immediately added and cell viability was determined after 18 hours (n=6/group from 3 separate experiments). ( b ) Cell viability was determined on INS-1 cells pretreated with sphingosine kinase inhibitor (2-( p -Hydroxyanilino)-4-( p -chlorophenyl) thiazole, 0.5 μM) or DMSO prior to delivery of adiponectin (3μg/mL) or PBS and maintained for 18 hours in the presence of 2% BSA or Palmitate (750 μM in 2% BSA) ( c ) Ceramidase activity was determined in lysates from cultured INS-1 cells under a range of pH conditions (n=4 from separate experiments) in the presence or absence of adiponectin (0.3 μg/mL, in vitro ). “Fold change over baseline” refers to the change compared to BSA treatment without adiponectin. ( d ) INS-1 cells were challenged with C2-ceramide (50 μM) in the presence or absence of S1P (5 μM) cell death was determined by live/dead staining with cFDA or annexin V (image is representative of 3 separate experiments, bar=50μm). ( e ) Apoptosis of INS-1 cells was determined by FACS analysis of annexin V and propidium iodide stained cells following 18 hours of treatment with BSA, palmitate (750 μM), or coadministered palmitate and S1P (5μM) (representative of 3 independent experiments). * denotes significant (p

    Journal: Nature medicine

    Article Title: The Pleiotropic Actions of Adiponectin are Initiated via Receptor-Mediated Activation of Ceramidase Activity

    doi: 10.1038/nm.2277

    Figure Lengend Snippet: Adiponectin alters sensitivity to ceramide-induced apoptosis in INS-1 β-cells ( a ) INS-1 cells were washed and removed to 2% BSA, Palmitate (750 μM in 2% BSA), or C2-Ceramide (50 μM in 2% BSA). Adiponectin (3 μg/mL) or PBS was immediately added and cell viability was determined after 18 hours (n=6/group from 3 separate experiments). ( b ) Cell viability was determined on INS-1 cells pretreated with sphingosine kinase inhibitor (2-( p -Hydroxyanilino)-4-( p -chlorophenyl) thiazole, 0.5 μM) or DMSO prior to delivery of adiponectin (3μg/mL) or PBS and maintained for 18 hours in the presence of 2% BSA or Palmitate (750 μM in 2% BSA) ( c ) Ceramidase activity was determined in lysates from cultured INS-1 cells under a range of pH conditions (n=4 from separate experiments) in the presence or absence of adiponectin (0.3 μg/mL, in vitro ). “Fold change over baseline” refers to the change compared to BSA treatment without adiponectin. ( d ) INS-1 cells were challenged with C2-ceramide (50 μM) in the presence or absence of S1P (5 μM) cell death was determined by live/dead staining with cFDA or annexin V (image is representative of 3 separate experiments, bar=50μm). ( e ) Apoptosis of INS-1 cells was determined by FACS analysis of annexin V and propidium iodide stained cells following 18 hours of treatment with BSA, palmitate (750 μM), or coadministered palmitate and S1P (5μM) (representative of 3 independent experiments). * denotes significant (p

    Article Snippet: Myriocin, fatty acid free BSA, palmitic acid, propidium iodide and Annexin VFITC were from Sigma.

    Techniques: Activity Assay, Cell Culture, In Vitro, Staining, FACS

    Effect of TGF-β on cardiomyocyte contractile function in myocytes isolated from FVB and metallothionein (MT) mice (maintained at normal temperature). A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal of relengthening (− dL/dt); E: Time-to-PS (TPS) and F: Time-to-90% relengthening (TR 90 ). Mean ± SEM, n = 73 – 80 cells from 3 mice per group, * p

    Journal: Free radical biology & medicine

    Article Title: Cardiac Overexpression of Metallothionein Rescues Cold Exposure-Induced Myocardial Contractile Dysfunction through Attenuation of Cardiac Fibrosis Despite Cardiomyocyte Mechanical Anomalies

    doi: 10.1016/j.freeradbiomed.2012.04.005

    Figure Lengend Snippet: Effect of TGF-β on cardiomyocyte contractile function in myocytes isolated from FVB and metallothionein (MT) mice (maintained at normal temperature). A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal of relengthening (− dL/dt); E: Time-to-PS (TPS) and F: Time-to-90% relengthening (TR 90 ). Mean ± SEM, n = 73 – 80 cells from 3 mice per group, * p

    Article Snippet: Mechanical properties of cardiomyocytes were assessed using an IonOptix™ soft-edge system (IonOptix, Milton, MA, USA).

    Techniques: Isolation, Mouse Assay

    Effect of metallothionein (MT) on ROS production and apoptosis in mice maintained at low temperature (LT, 4°C) for 3 months. A: Representative DCF fluorescent images (400×) showing ROS production in cardiomyocytes from FVB and MT mice with or without low temperature exposure; B: Pooled data of ROS production from 15 fields from 4 mice per group; C: Bax expression; and D: Bcl-xl expression. Insets: Representative gels depicting Bax and Bcl-xl expression using specific antibodies. GAPDH was used as the loading control; D: Assessment of apoptosis using TUNEL staining in myocardium. All nuclei were stained with DAPI shown in blue in the right column. The TUNEL-positive nuclei were visualized with fluorescein (green) in the left column. Original magnification = 400×. Quantified data are shown in panel F. Mean ± SEM, n = 3–5 mice (panels C–D) or 12 fields from 3 mice (panel F) per group, * p

    Journal: Free radical biology & medicine

    Article Title: Cardiac Overexpression of Metallothionein Rescues Cold Exposure-Induced Myocardial Contractile Dysfunction through Attenuation of Cardiac Fibrosis Despite Cardiomyocyte Mechanical Anomalies

    doi: 10.1016/j.freeradbiomed.2012.04.005

    Figure Lengend Snippet: Effect of metallothionein (MT) on ROS production and apoptosis in mice maintained at low temperature (LT, 4°C) for 3 months. A: Representative DCF fluorescent images (400×) showing ROS production in cardiomyocytes from FVB and MT mice with or without low temperature exposure; B: Pooled data of ROS production from 15 fields from 4 mice per group; C: Bax expression; and D: Bcl-xl expression. Insets: Representative gels depicting Bax and Bcl-xl expression using specific antibodies. GAPDH was used as the loading control; D: Assessment of apoptosis using TUNEL staining in myocardium. All nuclei were stained with DAPI shown in blue in the right column. The TUNEL-positive nuclei were visualized with fluorescein (green) in the left column. Original magnification = 400×. Quantified data are shown in panel F. Mean ± SEM, n = 3–5 mice (panels C–D) or 12 fields from 3 mice (panel F) per group, * p

    Article Snippet: Mechanical properties of cardiomyocytes were assessed using an IonOptix™ soft-edge system (IonOptix, Milton, MA, USA).

    Techniques: Mouse Assay, Expressing, TUNEL Assay, Staining

    Effect of heavy metal scavenger metallothionein (MT) on low temperature-induced cardiomyocyte contractile dysfunction. FVB and MT mice were kept at room temperature or 4°C for 3 months prior to the mechanical assessment. A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal of relengthening (− dL/dt); E: Time-to-PS (TPS) and F: Time-to-90% relengthening (TR 90 ). Mean ± SEM, n = 123 –126 cells from 4 mice per group, * p

    Journal: Free radical biology & medicine

    Article Title: Cardiac Overexpression of Metallothionein Rescues Cold Exposure-Induced Myocardial Contractile Dysfunction through Attenuation of Cardiac Fibrosis Despite Cardiomyocyte Mechanical Anomalies

    doi: 10.1016/j.freeradbiomed.2012.04.005

    Figure Lengend Snippet: Effect of heavy metal scavenger metallothionein (MT) on low temperature-induced cardiomyocyte contractile dysfunction. FVB and MT mice were kept at room temperature or 4°C for 3 months prior to the mechanical assessment. A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal of relengthening (− dL/dt); E: Time-to-PS (TPS) and F: Time-to-90% relengthening (TR 90 ). Mean ± SEM, n = 123 –126 cells from 4 mice per group, * p

    Article Snippet: Mechanical properties of cardiomyocytes were assessed using an IonOptix™ soft-edge system (IonOptix, Milton, MA, USA).

    Techniques: Mouse Assay

    Effect of heavy metal scavenger metallothionein (MT) on low temperature (LT, 4°C, 3 months)-induced change in intracellular Ca 2+ homeostasis in cardiomyocytes. FVB and MT mice were kept at room temperature or 4°C for 3 months prior to intracellular Ca 2+ assessment. A: Resting fura-2 fluorescence intensity (FFI); B: Electrically-stimulated rise in FFI (ΔFFI); C: Single exponential intracellular Ca 2+ decay rate; and D: Bi exponential intracellular Ca 2+ decay rate. Mean ± SEM, n = 59 – 60 cells from 4 mice per group, * p

    Journal: Free radical biology & medicine

    Article Title: Cardiac Overexpression of Metallothionein Rescues Cold Exposure-Induced Myocardial Contractile Dysfunction through Attenuation of Cardiac Fibrosis Despite Cardiomyocyte Mechanical Anomalies

    doi: 10.1016/j.freeradbiomed.2012.04.005

    Figure Lengend Snippet: Effect of heavy metal scavenger metallothionein (MT) on low temperature (LT, 4°C, 3 months)-induced change in intracellular Ca 2+ homeostasis in cardiomyocytes. FVB and MT mice were kept at room temperature or 4°C for 3 months prior to intracellular Ca 2+ assessment. A: Resting fura-2 fluorescence intensity (FFI); B: Electrically-stimulated rise in FFI (ΔFFI); C: Single exponential intracellular Ca 2+ decay rate; and D: Bi exponential intracellular Ca 2+ decay rate. Mean ± SEM, n = 59 – 60 cells from 4 mice per group, * p

    Article Snippet: Mechanical properties of cardiomyocytes were assessed using an IonOptix™ soft-edge system (IonOptix, Milton, MA, USA).

    Techniques: Mouse Assay, Fluorescence

    Rab3D release and actin coating of zymogen granules are interdependent events. ( a – c ) Double localization of rab3D and filamentous actin in tissue challenged with carbamylcholine (10 μM, 1 h). A close-up of part of an acinar lumen (asterisk) and the underlying zymogen granule field is shown. Rab3D immunofluorescence is depicted in a , and the corresponding phalloidin fluorescence is shown in b . The merge of the rab3D and phalloidin fluorescence images is shown in c . Note that the actin-coated granule (arrow) is not outlined by rab3D immunoreactivity, in contrast to the other zymogen granules. ( d ) Rab3D immunofluorescence in an acinus of tissue that was stimulated with carbamylcholine, showing absence of rab3D immunoreactivity on the apical membrane surrounding the lumen (asterisk). ( e ) Rab3D immunofluorescence in an acinus stimulated with carbamylcholine in the presence of cytochalasin D. The dramatically enlarged lumen (asterisks) is now outlined by strong rab3D staining. ( f – h ) Double localization of rab3D and filamentous actin in an acinus stimulated with carbamylcholine in the presence of latrunculin B and 2,3-butanedione monoxime. Phalloidin fluorescence is shown in ( f ). Note the presence of numerous actin-coated granules packed in clusters. Also note that the acinar lumen has not expanded. The corresponding rab3D immunofluorescence is depicted in g , and the merge between both fluorescence images is shown in h . Boxes in f and g outline an area that is shown at higher magnification in i (phalloidin), j (rab3D), and k (merge). Note that the actin-coated granules (two are indicated by arrows) are rab3D-immunoreactive. [Bars = 2 μm ( b , d , and i ) and 5 μm ( e and f ).]

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Actin coating of secretory granules during regulated exocytosis correlates with the release of rab3D

    doi:

    Figure Lengend Snippet: Rab3D release and actin coating of zymogen granules are interdependent events. ( a – c ) Double localization of rab3D and filamentous actin in tissue challenged with carbamylcholine (10 μM, 1 h). A close-up of part of an acinar lumen (asterisk) and the underlying zymogen granule field is shown. Rab3D immunofluorescence is depicted in a , and the corresponding phalloidin fluorescence is shown in b . The merge of the rab3D and phalloidin fluorescence images is shown in c . Note that the actin-coated granule (arrow) is not outlined by rab3D immunoreactivity, in contrast to the other zymogen granules. ( d ) Rab3D immunofluorescence in an acinus of tissue that was stimulated with carbamylcholine, showing absence of rab3D immunoreactivity on the apical membrane surrounding the lumen (asterisk). ( e ) Rab3D immunofluorescence in an acinus stimulated with carbamylcholine in the presence of cytochalasin D. The dramatically enlarged lumen (asterisks) is now outlined by strong rab3D staining. ( f – h ) Double localization of rab3D and filamentous actin in an acinus stimulated with carbamylcholine in the presence of latrunculin B and 2,3-butanedione monoxime. Phalloidin fluorescence is shown in ( f ). Note the presence of numerous actin-coated granules packed in clusters. Also note that the acinar lumen has not expanded. The corresponding rab3D immunofluorescence is depicted in g , and the merge between both fluorescence images is shown in h . Boxes in f and g outline an area that is shown at higher magnification in i (phalloidin), j (rab3D), and k (merge). Note that the actin-coated granules (two are indicated by arrows) are rab3D-immunoreactive. [Bars = 2 μm ( b , d , and i ) and 5 μm ( e and f ).]

    Article Snippet: Stimulation with carbamylcholine was carried out at 37°C and lasted for 1 h. In some experiments, jasplakinolide (3 μM; Molecular Probes), cytochalasin D (10 μM; Sigma), latrunculin B (1 μM; Alexis, San Diego, CA), or 2,3-butanedione monoxime (50 mM; Sigma) was administered, as indicated in Results and Discussion and the figure legends.

    Techniques: Immunofluorescence, Fluorescence, Staining

    Overexpression of FKBP12.6 significantly protects cardiomyocytes from AngII‐induced hypertrophy through inhibiting the Ca 2+ /CaMKII, AKT/GSK3β and AKT/mTOR signalling pathways in H9c2 cells. FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with PBS or with 200 nmol/L AngII for 20 min. The expressions of the total and phosphorylated CaMKII (A), AKT, GSK3β and mTOR (D) were detected by Western blot. The quantitative expressions of total and phosphoryated CaMKII, AKT and GSK3β protein were performed by density analysis, and the protein expression of p‐CaMKII (B), p‐AKT (C), p‐GSK3β (E) and p‐mTOR (F) was normalized to total CaMKII, AKT, GSK3β and mTOR. GAPDH was used as the loading controls. The data represent the mean ± SEM from three independent experiments, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro, et al. FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro

    doi: 10.1111/jcmm.13645

    Figure Lengend Snippet: Overexpression of FKBP12.6 significantly protects cardiomyocytes from AngII‐induced hypertrophy through inhibiting the Ca 2+ /CaMKII, AKT/GSK3β and AKT/mTOR signalling pathways in H9c2 cells. FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with PBS or with 200 nmol/L AngII for 20 min. The expressions of the total and phosphorylated CaMKII (A), AKT, GSK3β and mTOR (D) were detected by Western blot. The quantitative expressions of total and phosphoryated CaMKII, AKT and GSK3β protein were performed by density analysis, and the protein expression of p‐CaMKII (B), p‐AKT (C), p‐GSK3β (E) and p‐mTOR (F) was normalized to total CaMKII, AKT, GSK3β and mTOR. GAPDH was used as the loading controls. The data represent the mean ± SEM from three independent experiments, * P

    Article Snippet: The protein and mRNA levels of FKBP12.6 in Flag‐control cells were down‐regulated by 24% and 30%, respectively after AngII administration for 24 hours (Figure A‐C).

    Techniques: Over Expression, Western Blot, Expressing

    Cardiac‐specific overexpression of FKBP12.6 markedly inhibited AngII‐induced elevation of calcineurin, CaMKII and AKT activities in mice. The expressions of calcineurin (CaN) and MCIP1.4 (a highly sensitive readout for calcineurin activity) were detected by Western blot (A) and quantitatively determined by density analysis (B,C) in hearts from FKBP12.6 TG and WT mice that were subjected to 14 days of AngII or saline infusion. The expressions of the total or phosphorylated proteins including CaMKII, AKT and mTOR were detected by Western blot analysis (D‐F) and the protein expression of p‐CaMKII (G), p‐AKT (H) and p‐mTOR (I) was normalized to total CaMKII, AKT and mTOR in the hearts of the mice. GAPDH expression was used as loading control. The data represent the mean ± SEM, n = 5, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro, et al. FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro

    doi: 10.1111/jcmm.13645

    Figure Lengend Snippet: Cardiac‐specific overexpression of FKBP12.6 markedly inhibited AngII‐induced elevation of calcineurin, CaMKII and AKT activities in mice. The expressions of calcineurin (CaN) and MCIP1.4 (a highly sensitive readout for calcineurin activity) were detected by Western blot (A) and quantitatively determined by density analysis (B,C) in hearts from FKBP12.6 TG and WT mice that were subjected to 14 days of AngII or saline infusion. The expressions of the total or phosphorylated proteins including CaMKII, AKT and mTOR were detected by Western blot analysis (D‐F) and the protein expression of p‐CaMKII (G), p‐AKT (H) and p‐mTOR (I) was normalized to total CaMKII, AKT and mTOR in the hearts of the mice. GAPDH expression was used as loading control. The data represent the mean ± SEM, n = 5, * P

    Article Snippet: The protein and mRNA levels of FKBP12.6 in Flag‐control cells were down‐regulated by 24% and 30%, respectively after AngII administration for 24 hours (Figure A‐C).

    Techniques: Over Expression, Mouse Assay, Activity Assay, Western Blot, Expressing

    Overexpression of FKBP12.6 protects H9c2 cells from AngII‐induced hypertrophy. FKBP12.6 overexpressing stable H9c2 cell lines were prepared as described in the methods, and both FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with or without 200 nmol/L AngII for 24 h. The expressions of FKBP12.6 proteins were detected by Western blot (A) and quantitatively determined by density analysis (B). The mRNA expression of FKBP12.6 was also examined by qRT‐PCR (C). The cell sizes of FKBP12.6 overexpression (F12.6) and Flag‐control (Flag) H9c2 cells were detected by Phalloidin‐Tetramethylrhodamine staining (D, ×200), and the cell area was calculated by measuring at least 200 cells per slide using Image‐Pro Plus 6 software (E). The expressions of BNP protein (F G) and the mRNA expression of ANF (H), β‐MHC (I) and α‐MHC (J) were determined by Western blot analysis and qRT‐PCR, respectively. All results were presented from three independent experiments, and the data represent the mean ± SEM, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro, et al. FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro

    doi: 10.1111/jcmm.13645

    Figure Lengend Snippet: Overexpression of FKBP12.6 protects H9c2 cells from AngII‐induced hypertrophy. FKBP12.6 overexpressing stable H9c2 cell lines were prepared as described in the methods, and both FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with or without 200 nmol/L AngII for 24 h. The expressions of FKBP12.6 proteins were detected by Western blot (A) and quantitatively determined by density analysis (B). The mRNA expression of FKBP12.6 was also examined by qRT‐PCR (C). The cell sizes of FKBP12.6 overexpression (F12.6) and Flag‐control (Flag) H9c2 cells were detected by Phalloidin‐Tetramethylrhodamine staining (D, ×200), and the cell area was calculated by measuring at least 200 cells per slide using Image‐Pro Plus 6 software (E). The expressions of BNP protein (F G) and the mRNA expression of ANF (H), β‐MHC (I) and α‐MHC (J) were determined by Western blot analysis and qRT‐PCR, respectively. All results were presented from three independent experiments, and the data represent the mean ± SEM, * P

    Article Snippet: The protein and mRNA levels of FKBP12.6 in Flag‐control cells were down‐regulated by 24% and 30%, respectively after AngII administration for 24 hours (Figure A‐C).

    Techniques: Over Expression, Western Blot, Expressing, Quantitative RT-PCR, Staining, Software

    FKBP12.6 deficiency aggravates AngII‐induced cardiac hypertrophy in vivo. FKBP12.6 −/− and WT mice were subjected to 14 d AngII or saline infusion, and then the hearts were isolated from the mice for histological and biochemical analyses. The mRNA expressions of FKBP12.6 in hearts from WT mice after AngII or saline infusion were determined by qRT‐PCR (A). The heart weight and body weight of each mouse were measured for calculating the ratio of heart weight/body weight (HW/BW) (B). The myocyte cross‐sectional areas were stained with FITC‐conjugated wheat germ agglutinin (C), and quantitatively analysed (D, ×400, n = 200 cells per section). The mRNA expressions of hypertrophic makers ANF (E) and BNP (F) were analysed by qRT‐PCR. The data represent the mean ± SEM, n = 5 mice, $ P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro, et al. FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro

    doi: 10.1111/jcmm.13645

    Figure Lengend Snippet: FKBP12.6 deficiency aggravates AngII‐induced cardiac hypertrophy in vivo. FKBP12.6 −/− and WT mice were subjected to 14 d AngII or saline infusion, and then the hearts were isolated from the mice for histological and biochemical analyses. The mRNA expressions of FKBP12.6 in hearts from WT mice after AngII or saline infusion were determined by qRT‐PCR (A). The heart weight and body weight of each mouse were measured for calculating the ratio of heart weight/body weight (HW/BW) (B). The myocyte cross‐sectional areas were stained with FITC‐conjugated wheat germ agglutinin (C), and quantitatively analysed (D, ×400, n = 200 cells per section). The mRNA expressions of hypertrophic makers ANF (E) and BNP (F) were analysed by qRT‐PCR. The data represent the mean ± SEM, n = 5 mice, $ P

    Article Snippet: The protein and mRNA levels of FKBP12.6 in Flag‐control cells were down‐regulated by 24% and 30%, respectively after AngII administration for 24 hours (Figure A‐C).

    Techniques: In Vivo, Mouse Assay, Isolation, Quantitative RT-PCR, Staining

    Overexpression of FKBP12.6 attenuates AngII‐induced myocardial apoptosis in H9c2 cells. FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with PBS or 200 nmol/L AngII for 20 min. The intracellular ROS productions were measured by a flow cytometry stained with CM‐H2DCFDA (A) and the mean DCF‐fluorescence intensity in different group of cells was quantitatively determined (B). Cell apoptosis was detected by a flow cytometry with annexin V‐FITC/propidium iodide (PI) double staining. Representative flow cytometric dot plots ( x ‐axis: annexin V staining/ y ‐axis: PI staining) show two‐cell populations: annexin V/PI‐negative cells (normal cells, lower left quadrant) and annexin V‐positive/PI‐negative cells (early apoptotic cells, lower right quadrant) and annexin V‐positive/PI‐negative cells(late apoptotic cells, lower right quadrant) (C). The annexin V‐positive/PI‐negative cells (apoptosis cells) are shown in (D). The expressions of Bax and Bcl2 proteins were detected by Western blot (E) in FKBP12.6 overexpression (F12.6) and Flag‐control (Flag) H9c2 cells after treated with AngII (200 nmol/L) for 24 h, and the Bax/Bcl2 ratios were quantitatively determined by density analysis (F). GAPDH was used as an internal control. The data represent the mean ± SEM from three independent experiments, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro, et al. FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro

    doi: 10.1111/jcmm.13645

    Figure Lengend Snippet: Overexpression of FKBP12.6 attenuates AngII‐induced myocardial apoptosis in H9c2 cells. FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with PBS or 200 nmol/L AngII for 20 min. The intracellular ROS productions were measured by a flow cytometry stained with CM‐H2DCFDA (A) and the mean DCF‐fluorescence intensity in different group of cells was quantitatively determined (B). Cell apoptosis was detected by a flow cytometry with annexin V‐FITC/propidium iodide (PI) double staining. Representative flow cytometric dot plots ( x ‐axis: annexin V staining/ y ‐axis: PI staining) show two‐cell populations: annexin V/PI‐negative cells (normal cells, lower left quadrant) and annexin V‐positive/PI‐negative cells (early apoptotic cells, lower right quadrant) and annexin V‐positive/PI‐negative cells(late apoptotic cells, lower right quadrant) (C). The annexin V‐positive/PI‐negative cells (apoptosis cells) are shown in (D). The expressions of Bax and Bcl2 proteins were detected by Western blot (E) in FKBP12.6 overexpression (F12.6) and Flag‐control (Flag) H9c2 cells after treated with AngII (200 nmol/L) for 24 h, and the Bax/Bcl2 ratios were quantitatively determined by density analysis (F). GAPDH was used as an internal control. The data represent the mean ± SEM from three independent experiments, * P

    Article Snippet: The protein and mRNA levels of FKBP12.6 in Flag‐control cells were down‐regulated by 24% and 30%, respectively after AngII administration for 24 hours (Figure A‐C).

    Techniques: Over Expression, Flow Cytometry, Cytometry, Staining, Fluorescence, Double Staining, Western Blot

    Overexpression of FKBP12.6 significantly reduces the elevation of the intracellular Ca 2+ concentrations ([Ca 2+ ]i) and inhibits the activities of Ca 2+ /calcineurin signalling pathways after AngII stimulation in H9c2 cells. The AngII‐induced elevation of the intracellular Ca 2+ was detected by flow cytometric analysis in FKBP12.6 overexpression (F12.6) and Flag‐control (Flag) H9c2 cells with Fluo3‐AM staining after 20‐min PBS or AngII treatment (A) and quantitatively determined by measuring the mean fluorescence intensity of Fluo‐3 in each group (B). FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with or without 200 nmol/L AngII for 24 h. The expressions of calcineurin (CaN) and MCIP1.4 proteins were detected by Western blot (C) and quantitatively determined by density analysis (D and E), respectively. The NFATc4 protein levels in cytosolic and nuclear fractions were analysed by Western blot (F). TATA‐binding protein (TBP), a nuclear protein marker, and Tubulin, a cytosolic protein marker, were used as an internal control, respectively. The contents of NFATc4 protein in nuclei (G) and cytosole (H) were quantitatively determined by density analysis. The data represent the mean ± SEM from three independent experiments, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro, et al. FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro

    doi: 10.1111/jcmm.13645

    Figure Lengend Snippet: Overexpression of FKBP12.6 significantly reduces the elevation of the intracellular Ca 2+ concentrations ([Ca 2+ ]i) and inhibits the activities of Ca 2+ /calcineurin signalling pathways after AngII stimulation in H9c2 cells. The AngII‐induced elevation of the intracellular Ca 2+ was detected by flow cytometric analysis in FKBP12.6 overexpression (F12.6) and Flag‐control (Flag) H9c2 cells with Fluo3‐AM staining after 20‐min PBS or AngII treatment (A) and quantitatively determined by measuring the mean fluorescence intensity of Fluo‐3 in each group (B). FKBP12.6 overexpressing (F12.6) and Flag‐control (Flag) H9c2 cells were treated with or without 200 nmol/L AngII for 24 h. The expressions of calcineurin (CaN) and MCIP1.4 proteins were detected by Western blot (C) and quantitatively determined by density analysis (D and E), respectively. The NFATc4 protein levels in cytosolic and nuclear fractions were analysed by Western blot (F). TATA‐binding protein (TBP), a nuclear protein marker, and Tubulin, a cytosolic protein marker, were used as an internal control, respectively. The contents of NFATc4 protein in nuclei (G) and cytosole (H) were quantitatively determined by density analysis. The data represent the mean ± SEM from three independent experiments, * P

    Article Snippet: The protein and mRNA levels of FKBP12.6 in Flag‐control cells were down‐regulated by 24% and 30%, respectively after AngII administration for 24 hours (Figure A‐C).

    Techniques: Over Expression, Flow Cytometry, Staining, Fluorescence, Western Blot, Binding Assay, Marker

    Cardiac‐specific overexpression of FKBP12.6 protects hearts from AngII‐induced cardiac hypertrophy in vivo. The expressions of FKBP12.6 protein in the hearts from three lines 2‐month‐old FKBP12.6 transgenic mice (FKBP12.6 TG) and their WT littermates were determined by Western blot (A). The heart weight and body weight of each mouse were measured for calculating the ratio of heart weight/body weight (HW/BW) in FKBP12.6 TG and WT mice which were subjected to 14 days AngII or saline infusion (B). The myocyte cross‐sectional areas were determined by FITC‐conjugated wheat germ agglutinin staining (D, upper, ×400) and quantitative analysis (C, n = 200 cells per section), and the histological examination was analysed by HE staining (D, lower, ×40). The expressions of BNP protein (E F) and the mRNA expressions of hypertrophic makers BNP (G) and ANF (H) in hearts from FKBP12.6 TG and WT mice after AngII or saline infusion were analysed by Western blot and qRT‐PCR. The data represent the mean ± SEM, n = 5, * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro, et al. FKBP12.6 protects heart from AngII‐induced hypertrophy through inhibiting Ca2+/calmodulin‐mediated signalling pathways in vivo and in vitro

    doi: 10.1111/jcmm.13645

    Figure Lengend Snippet: Cardiac‐specific overexpression of FKBP12.6 protects hearts from AngII‐induced cardiac hypertrophy in vivo. The expressions of FKBP12.6 protein in the hearts from three lines 2‐month‐old FKBP12.6 transgenic mice (FKBP12.6 TG) and their WT littermates were determined by Western blot (A). The heart weight and body weight of each mouse were measured for calculating the ratio of heart weight/body weight (HW/BW) in FKBP12.6 TG and WT mice which were subjected to 14 days AngII or saline infusion (B). The myocyte cross‐sectional areas were determined by FITC‐conjugated wheat germ agglutinin staining (D, upper, ×400) and quantitative analysis (C, n = 200 cells per section), and the histological examination was analysed by HE staining (D, lower, ×40). The expressions of BNP protein (E F) and the mRNA expressions of hypertrophic makers BNP (G) and ANF (H) in hearts from FKBP12.6 TG and WT mice after AngII or saline infusion were analysed by Western blot and qRT‐PCR. The data represent the mean ± SEM, n = 5, * P

    Article Snippet: The protein and mRNA levels of FKBP12.6 in Flag‐control cells were down‐regulated by 24% and 30%, respectively after AngII administration for 24 hours (Figure A‐C).

    Techniques: Over Expression, In Vivo, Transgenic Assay, Mouse Assay, Western Blot, Staining, Quantitative RT-PCR

    AAV9.CXCR4 dose response in vivo . AAV9.CXCR4 WT was injected via the tail vein with increasing viral genomes/mouse, ranging from 2e9 to 3e11. Hearts were harvested four weeks post-injection. (A) CXCR4 mRNA was quantified by RT-PCR. (B) CXCR4 protein expression was assessed by western blot. (C) Immunostaining of CXCR4 in the heart indicated proper membrane localization.

    Journal: Journal of molecular and cellular cardiology

    Article Title: CXCR4 gene transfer prevents pressure overload induced heart failure

    doi: 10.1016/j.yjmcc.2012.05.016

    Figure Lengend Snippet: AAV9.CXCR4 dose response in vivo . AAV9.CXCR4 WT was injected via the tail vein with increasing viral genomes/mouse, ranging from 2e9 to 3e11. Hearts were harvested four weeks post-injection. (A) CXCR4 mRNA was quantified by RT-PCR. (B) CXCR4 protein expression was assessed by western blot. (C) Immunostaining of CXCR4 in the heart indicated proper membrane localization.

    Article Snippet: AAV9.CXCR4 treatment prevented calcineurin activity at TAC 2weeks further supporting the anti-hypertrophic capability of CXCR4.

    Techniques: In Vivo, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Immunostaining

    VEGF expression during pressure overload. (A) Cardiac sections were stained with isolectin-488 to identify myocardial capillary density. (B) Quantitative real time PCR data demonstrates an increase in VEGF transcription in TAC+AAV.CXCR4 group. (n=4 mice/group *= p

    Journal: Journal of molecular and cellular cardiology

    Article Title: CXCR4 gene transfer prevents pressure overload induced heart failure

    doi: 10.1016/j.yjmcc.2012.05.016

    Figure Lengend Snippet: VEGF expression during pressure overload. (A) Cardiac sections were stained with isolectin-488 to identify myocardial capillary density. (B) Quantitative real time PCR data demonstrates an increase in VEGF transcription in TAC+AAV.CXCR4 group. (n=4 mice/group *= p

    Article Snippet: AAV9.CXCR4 treatment prevented calcineurin activity at TAC 2weeks further supporting the anti-hypertrophic capability of CXCR4.

    Techniques: Expressing, Staining, Real-time Polymerase Chain Reaction, Mouse Assay

    CXCL12/CXCR4 expression during cardiac myocyte hypertrophy in vitro . (A) Isolated adult rat cardiac myocytes were treated with isoproterenol (500nM) for 24 hours to induce cellular hypertrophy. Myocytes were immunostained for CXCR4 pre- and post-isoproterenol treatment. Additionally, RNA was harvested from the cell lysates and CXCL12/CXCR4 mRNA content was quantified. (n=4 independent experiments, * p

    Journal: Journal of molecular and cellular cardiology

    Article Title: CXCR4 gene transfer prevents pressure overload induced heart failure

    doi: 10.1016/j.yjmcc.2012.05.016

    Figure Lengend Snippet: CXCL12/CXCR4 expression during cardiac myocyte hypertrophy in vitro . (A) Isolated adult rat cardiac myocytes were treated with isoproterenol (500nM) for 24 hours to induce cellular hypertrophy. Myocytes were immunostained for CXCR4 pre- and post-isoproterenol treatment. Additionally, RNA was harvested from the cell lysates and CXCL12/CXCR4 mRNA content was quantified. (n=4 independent experiments, * p

    Article Snippet: AAV9.CXCR4 treatment prevented calcineurin activity at TAC 2weeks further supporting the anti-hypertrophic capability of CXCR4.

    Techniques: Expressing, In Vitro, Isolation

    (A) Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L) and NAM (5 and 10 mmol/L). THP-1 cells were treated with LPS (10 μg/mL) for 4, 8, 12 h. SIRT1 activity was determined by colorimetric assay. (*Represents P ≤ 0.05 LPS exposed cells in the absence or presence of DIQ; # represents P ≤ 0.05 LPS exposed cells treated with DIQ or DIQ and NAM.) Assay shown is representative of three experiments with similar results. (B) Representative autoradiograph of Western blot analysis for nuclear SIRT1 concentration in THP-1 cells treated with LPS in the presence or absence of DIQ (100 and 300 μmol/L). THP-1 cells were treated with LPS (10 μg/mL) for 4 and 8 h. The gel is representative of three experiments with similar results. (C) Nuclear proteins were extracted from WT and PARP1 −/− cells and SIRT1 activity was determined by colorimetric assay. (*Represents P ≤ 0.05 versus WT cells.) (D) Representative autoradiograph of Western blot analysis for supernatant HMGB1 levels in THP-1 cells treated with LPS in the presence or absence of SRT1720 (0.01 and 0.1 μmol/L), a SIRT1 activator. THP-1 cells were treated with LPS (10 μg/mL) for 18 h. The gel is representative of three experiments with similar results.

    Journal: Molecular Medicine

    Article Title: Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    doi: 10.2119/molmed.2014.00156

    Figure Lengend Snippet: (A) Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L) and NAM (5 and 10 mmol/L). THP-1 cells were treated with LPS (10 μg/mL) for 4, 8, 12 h. SIRT1 activity was determined by colorimetric assay. (*Represents P ≤ 0.05 LPS exposed cells in the absence or presence of DIQ; # represents P ≤ 0.05 LPS exposed cells treated with DIQ or DIQ and NAM.) Assay shown is representative of three experiments with similar results. (B) Representative autoradiograph of Western blot analysis for nuclear SIRT1 concentration in THP-1 cells treated with LPS in the presence or absence of DIQ (100 and 300 μmol/L). THP-1 cells were treated with LPS (10 μg/mL) for 4 and 8 h. The gel is representative of three experiments with similar results. (C) Nuclear proteins were extracted from WT and PARP1 −/− cells and SIRT1 activity was determined by colorimetric assay. (*Represents P ≤ 0.05 versus WT cells.) (D) Representative autoradiograph of Western blot analysis for supernatant HMGB1 levels in THP-1 cells treated with LPS in the presence or absence of SRT1720 (0.01 and 0.1 μmol/L), a SIRT1 activator. THP-1 cells were treated with LPS (10 μg/mL) for 18 h. The gel is representative of three experiments with similar results.

    Article Snippet: However, no similar posttranslational modification has been seen on SIRT1 by PARP1 in response to DNA damage ( ).

    Techniques: Activity Assay, Colorimetric Assay, Autoradiography, Western Blot, Concentration Assay

    (A) Representative confocal images of proximity ligation assay (PLA). Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 6 h. PLA amplification corresponds with the interaction of PARP1 with SIRT1 and is visualized as red-pink spots localized mainly in the nucleus. (B) Coimmunoprecipitation analysis from THP-1 cell lysates. Panel 1: Samples were immunoprecipitated with anti-SIRT1 and immunoblotted with anti-poly(ADP-ribose). The blot was then stripped and reprobed for SIRT1 (panel 2).

    Journal: Molecular Medicine

    Article Title: Poly(ADP-Ribose) Polymerase 1–Sirtuin 1 Functional Interplay Regulates LPS-Mediated High Mobility Group Box 1 Secretion

    doi: 10.2119/molmed.2014.00156

    Figure Lengend Snippet: (A) Representative confocal images of proximity ligation assay (PLA). Nuclear proteins were extracted from THP-1 cells treated with LPS in the presence or absence of DIQ (300 μmol/L), a PARP1 inhibitor. THP-1 cells were treated with LPS (10 μg/mL) for 6 h. PLA amplification corresponds with the interaction of PARP1 with SIRT1 and is visualized as red-pink spots localized mainly in the nucleus. (B) Coimmunoprecipitation analysis from THP-1 cell lysates. Panel 1: Samples were immunoprecipitated with anti-SIRT1 and immunoblotted with anti-poly(ADP-ribose). The blot was then stripped and reprobed for SIRT1 (panel 2).

    Article Snippet: However, no similar posttranslational modification has been seen on SIRT1 by PARP1 in response to DNA damage ( ).

    Techniques: Proximity Ligation Assay, Amplification, Immunoprecipitation

    Autophagy inhibition attenuates FoxO1-induced reduction in cell size and relative protein content. Cultured cardiomyocytes were transduced with Ad-FoxO1-WT or Ad-LacZ for 24 hours or Ad-sh-Beclin1 for 96 hours, and treated with 10 mM 3-methyladenine (3-MA) for 24 hours. A) Representative immunoblots. B) Densitometric analyses. C) Representative images of cardiomyocytes viewed under a light microscope. D) Relative cardiomyocyte cross-sectional area. E) Relative protein content. Data represent means from at least 6 different myocyte cultures. * p

    Journal: PLoS ONE

    Article Title: Autophagy Plays an Essential Role in Mediating Regression of Hypertrophy during Unloading of the Heart

    doi: 10.1371/journal.pone.0051632

    Figure Lengend Snippet: Autophagy inhibition attenuates FoxO1-induced reduction in cell size and relative protein content. Cultured cardiomyocytes were transduced with Ad-FoxO1-WT or Ad-LacZ for 24 hours or Ad-sh-Beclin1 for 96 hours, and treated with 10 mM 3-methyladenine (3-MA) for 24 hours. A) Representative immunoblots. B) Densitometric analyses. C) Representative images of cardiomyocytes viewed under a light microscope. D) Relative cardiomyocyte cross-sectional area. E) Relative protein content. Data represent means from at least 6 different myocyte cultures. * p

    Article Snippet: Discussion We here show that a) regression of cardiac hypertrophy is induced by left ventricular unloading and de-stretch of cultured cardiomyocytes after mechanical stretch, and is accompanied by increased autophagy and upregulation of FoxO1, b) FoxO1 increases the expression of autophagy genes and autophagosome formation in mouse hearts in vivo , c) overexpression of FoxO1 reduces cardiomyocyte size, whereas inhibition of autophagy attenuates FoxO1-induced reductions in cell size and protein content, and d) autophagy and FoxO1 are required to mediate regression of cardiac hypertrophy both in vitro and in vivo.

    Techniques: Inhibition, Cell Culture, Transduction, Western Blot, Light Microscopy

    FoxO1 expression and autophagy are increased in an in vitro model of regression of cardiac hypertrophy. Cardiomyocytes were cultured in BioFlex plates, subjected to mechanical cyclic stretch for 36 hours (Stretch) and incubated without stretch for 36 hours (de-stretch). A) Scheme showing the regimen of mechanical stretch and de-stretch. B) Relative protein content. C–D) mRNA levels of ANF and FoxO1, determined by qRT-PCR. E) Representative immunoblots. F) Densitometric analyses. Data represent means from at least 4 different myocyte cultures. * p

    Journal: PLoS ONE

    Article Title: Autophagy Plays an Essential Role in Mediating Regression of Hypertrophy during Unloading of the Heart

    doi: 10.1371/journal.pone.0051632

    Figure Lengend Snippet: FoxO1 expression and autophagy are increased in an in vitro model of regression of cardiac hypertrophy. Cardiomyocytes were cultured in BioFlex plates, subjected to mechanical cyclic stretch for 36 hours (Stretch) and incubated without stretch for 36 hours (de-stretch). A) Scheme showing the regimen of mechanical stretch and de-stretch. B) Relative protein content. C–D) mRNA levels of ANF and FoxO1, determined by qRT-PCR. E) Representative immunoblots. F) Densitometric analyses. Data represent means from at least 4 different myocyte cultures. * p

    Article Snippet: Discussion We here show that a) regression of cardiac hypertrophy is induced by left ventricular unloading and de-stretch of cultured cardiomyocytes after mechanical stretch, and is accompanied by increased autophagy and upregulation of FoxO1, b) FoxO1 increases the expression of autophagy genes and autophagosome formation in mouse hearts in vivo , c) overexpression of FoxO1 reduces cardiomyocyte size, whereas inhibition of autophagy attenuates FoxO1-induced reductions in cell size and protein content, and d) autophagy and FoxO1 are required to mediate regression of cardiac hypertrophy both in vitro and in vivo.

    Techniques: Expressing, In Vitro, Cell Culture, Incubation, Quantitative RT-PCR, Western Blot

    Autophagy and FoxO1 expression are upregulated during regression of cardiac hypertrophy. Control C57BL/6 and Tg-GFP-LC3 mice were subjected to sham, 1W TAC and 1W DeTAC surgeries. A) Representative immunoblots of autophagy markers and FoxO1 from mouse hearts. B) Densitometric analyses. C) Representative images of fluorescent LC3 puncta in hearts from Tg-GFP-LC3 mice. Arrows indicate LC3 puncta. D) Mean number of GFP-LC3 dots/cell. Data represent means from at least 3 mice each. * p

    Journal: PLoS ONE

    Article Title: Autophagy Plays an Essential Role in Mediating Regression of Hypertrophy during Unloading of the Heart

    doi: 10.1371/journal.pone.0051632

    Figure Lengend Snippet: Autophagy and FoxO1 expression are upregulated during regression of cardiac hypertrophy. Control C57BL/6 and Tg-GFP-LC3 mice were subjected to sham, 1W TAC and 1W DeTAC surgeries. A) Representative immunoblots of autophagy markers and FoxO1 from mouse hearts. B) Densitometric analyses. C) Representative images of fluorescent LC3 puncta in hearts from Tg-GFP-LC3 mice. Arrows indicate LC3 puncta. D) Mean number of GFP-LC3 dots/cell. Data represent means from at least 3 mice each. * p

    Article Snippet: Discussion We here show that a) regression of cardiac hypertrophy is induced by left ventricular unloading and de-stretch of cultured cardiomyocytes after mechanical stretch, and is accompanied by increased autophagy and upregulation of FoxO1, b) FoxO1 increases the expression of autophagy genes and autophagosome formation in mouse hearts in vivo , c) overexpression of FoxO1 reduces cardiomyocyte size, whereas inhibition of autophagy attenuates FoxO1-induced reductions in cell size and protein content, and d) autophagy and FoxO1 are required to mediate regression of cardiac hypertrophy both in vitro and in vivo.

    Techniques: Expressing, Mouse Assay, Western Blot

    Overexpression of FoxO1 regulates autophagy in vivo. Transgenic mice with cardiac-specific overexpression of WT-FoxO1 (Tg-FoxO1) were generated. A) Representative immunoblots comparing FoxO1 expression levels in the two lines (lines #8 and #36) of Tg-FoxO1 and nontransgenic (NTg) mice. B) Densitometric analyses. C) mRNA levels of autophagy genes Gabarapl1, Bnip3 and Ulk2. D) Representative immunoblots of autophagy markers. E) Densitometric analyses. F–G) Tg-FoxO1 mice were bred with Tg-GFP-LC3 to generate Tg-GFP-LC3 – FoxO1 bigenic mice. F) Representative images of GFP-LC3 puncta. Arrows indicate LC3 puncta. G) Mean number of GFP-LC3 dots/cell. Data represent means from at least 4 individual mice. * p

    Journal: PLoS ONE

    Article Title: Autophagy Plays an Essential Role in Mediating Regression of Hypertrophy during Unloading of the Heart

    doi: 10.1371/journal.pone.0051632

    Figure Lengend Snippet: Overexpression of FoxO1 regulates autophagy in vivo. Transgenic mice with cardiac-specific overexpression of WT-FoxO1 (Tg-FoxO1) were generated. A) Representative immunoblots comparing FoxO1 expression levels in the two lines (lines #8 and #36) of Tg-FoxO1 and nontransgenic (NTg) mice. B) Densitometric analyses. C) mRNA levels of autophagy genes Gabarapl1, Bnip3 and Ulk2. D) Representative immunoblots of autophagy markers. E) Densitometric analyses. F–G) Tg-FoxO1 mice were bred with Tg-GFP-LC3 to generate Tg-GFP-LC3 – FoxO1 bigenic mice. F) Representative images of GFP-LC3 puncta. Arrows indicate LC3 puncta. G) Mean number of GFP-LC3 dots/cell. Data represent means from at least 4 individual mice. * p

    Article Snippet: Discussion We here show that a) regression of cardiac hypertrophy is induced by left ventricular unloading and de-stretch of cultured cardiomyocytes after mechanical stretch, and is accompanied by increased autophagy and upregulation of FoxO1, b) FoxO1 increases the expression of autophagy genes and autophagosome formation in mouse hearts in vivo , c) overexpression of FoxO1 reduces cardiomyocyte size, whereas inhibition of autophagy attenuates FoxO1-induced reductions in cell size and protein content, and d) autophagy and FoxO1 are required to mediate regression of cardiac hypertrophy both in vitro and in vivo.

    Techniques: Over Expression, In Vivo, Transgenic Assay, Mouse Assay, Generated, Western Blot, Expressing

    FoxO1 and autophagy are required for regression of cardiac hypertrophy. Control C57BL/6 mice and mice with heterozygous knockout of Beclin1 ( beclin1+/− ) were subjected to 1W TAC and 1W DeTAC surgeries. A) Representative immunoblots. B) Densitometric analyses. C) Left ventricular weight/body weight (LVW/BW). D) Cultured cardiomyocytes were transduced with Ad-sh-Scr, Ad-sh-Beclin1 and Ad-sh-FoxO1 and subjected to mechanical stretch/de-stretch. The percentage reduction in relative protein content after de-stretch compared to after stretch is represented as % regression of cardiac hypertrophy in vitro . E) A scheme showing the proposed hypothesis of this study. * p

    Journal: PLoS ONE

    Article Title: Autophagy Plays an Essential Role in Mediating Regression of Hypertrophy during Unloading of the Heart

    doi: 10.1371/journal.pone.0051632

    Figure Lengend Snippet: FoxO1 and autophagy are required for regression of cardiac hypertrophy. Control C57BL/6 mice and mice with heterozygous knockout of Beclin1 ( beclin1+/− ) were subjected to 1W TAC and 1W DeTAC surgeries. A) Representative immunoblots. B) Densitometric analyses. C) Left ventricular weight/body weight (LVW/BW). D) Cultured cardiomyocytes were transduced with Ad-sh-Scr, Ad-sh-Beclin1 and Ad-sh-FoxO1 and subjected to mechanical stretch/de-stretch. The percentage reduction in relative protein content after de-stretch compared to after stretch is represented as % regression of cardiac hypertrophy in vitro . E) A scheme showing the proposed hypothesis of this study. * p

    Article Snippet: Discussion We here show that a) regression of cardiac hypertrophy is induced by left ventricular unloading and de-stretch of cultured cardiomyocytes after mechanical stretch, and is accompanied by increased autophagy and upregulation of FoxO1, b) FoxO1 increases the expression of autophagy genes and autophagosome formation in mouse hearts in vivo , c) overexpression of FoxO1 reduces cardiomyocyte size, whereas inhibition of autophagy attenuates FoxO1-induced reductions in cell size and protein content, and d) autophagy and FoxO1 are required to mediate regression of cardiac hypertrophy both in vitro and in vivo.

    Techniques: Mouse Assay, Knock-Out, Western Blot, Cell Culture, Transduction, In Vitro

    Cardiac SGLT1 expression was regulated by AMPK. (A) Levels of Thr 172 phosphorylated AMPK a subunit (P-AMPKα), which reflect AMPK activity, were increased in 8 week old male WT FVB mice after administration of the AMPK activator AICAR (500 μg/kg IP, twice at a 3 h interval). (B) Concurrently, cardiac SGLT1 mRNA expression was increased in these mice after administration of AICAR ( n = 6 / group). (C) Levels of Thr 172 phosphorylated AMPK α subunit (P-AMPKα) were normalized in TG T400N /TG α2DN relative to TG T400N hearts at age 2 weeks. (D) Double transgenic mice (TG T400N /TG α2DN ) exhibited attenuation of cardiac glucose uptake ( n = 3 / group). (E) Increased SGLT1 mRNA expression in TG T00N hearts was attenuated in TG T400N /TG α2DN hearts, as assessed by QPCR ( n = 3 / group). (F) An immunoblot showed that the increased SGLT1 protein expression observed in TG T00N hearts was attenuated in TG T400N /TG α2DN hearts. (G) Densitometry analysis of the immunoblot shown in panel F. Chromatin immunoprecipitation (ChIP) showed that increased expression of SGLT1 in TG T00N hearts relative to WT was associated with increased binding of (H) HNF-1 and (I) Sp1 to the promoter of the SLC5A1 gene encoding SGLT1 ( n = 3 / group). Molecular weights (MW) on immunoblots were estimated by protein marker sizes. Coomassie blue staining was used to document the relative quantity of protein loaded for the immunoblots. *, P

    Journal: Journal of molecular and cellular cardiology

    Article Title: SGLT1, a Novel Cardiac Glucose Transporter, Mediates Increased Glucose Uptake in PRKAG2 Cardiomyopathy

    doi: 10.1016/j.yjmcc.2010.06.003

    Figure Lengend Snippet: Cardiac SGLT1 expression was regulated by AMPK. (A) Levels of Thr 172 phosphorylated AMPK a subunit (P-AMPKα), which reflect AMPK activity, were increased in 8 week old male WT FVB mice after administration of the AMPK activator AICAR (500 μg/kg IP, twice at a 3 h interval). (B) Concurrently, cardiac SGLT1 mRNA expression was increased in these mice after administration of AICAR ( n = 6 / group). (C) Levels of Thr 172 phosphorylated AMPK α subunit (P-AMPKα) were normalized in TG T400N /TG α2DN relative to TG T400N hearts at age 2 weeks. (D) Double transgenic mice (TG T400N /TG α2DN ) exhibited attenuation of cardiac glucose uptake ( n = 3 / group). (E) Increased SGLT1 mRNA expression in TG T00N hearts was attenuated in TG T400N /TG α2DN hearts, as assessed by QPCR ( n = 3 / group). (F) An immunoblot showed that the increased SGLT1 protein expression observed in TG T00N hearts was attenuated in TG T400N /TG α2DN hearts. (G) Densitometry analysis of the immunoblot shown in panel F. Chromatin immunoprecipitation (ChIP) showed that increased expression of SGLT1 in TG T00N hearts relative to WT was associated with increased binding of (H) HNF-1 and (I) Sp1 to the promoter of the SLC5A1 gene encoding SGLT1 ( n = 3 / group). Molecular weights (MW) on immunoblots were estimated by protein marker sizes. Coomassie blue staining was used to document the relative quantity of protein loaded for the immunoblots. *, P

    Article Snippet: Thus, there may be differences between acute and chronic AMPK regulation of SGLT1, and differences among tissues, cell types, and possibly species.

    Techniques: Expressing, Activity Assay, Mouse Assay, Transgenic Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay, Western Blot, Marker, Staining

    Cardiac GLUT1, GLUT4, and SGLT1 mRNA and protein expression in TG T400N mice. (A) Total cardiac GLUT1 and GLUT4 transcript expression, as assessed by QPCR, were similar in TG T400N mice and WT littermates at ages 2 and 8 weeks, whereas cardiac SGLT1 transcript expression was increased in TG T400N mice at ages 2 and 8 weeks ( n = 3 / group). (B) In whole heart homogenates, densitometry analysis of immunoblots showed that GLUT1 protein was significantly increased, GLUT4 protein was significantly decreased, and SGLT1 protein was unchanged in TG T400N mice relative to WT littermates at ages 2 and 8 weeks. (C) In total membrane extracts, densitometry analysis of immunoblots showed that GLUT1 and GLUT4 protein expression was not significantly different in TG T400N mice relative to WT littermates at ages 2 and 8 weeks, whereas SGLT1 protein expression was significantly increased in TG T400N mice at ages 2 and 8 weeks. (D) In sarcolemmal membrane extracts, GLUT1 and SGLT1 protein expression appeared to be mildly increased at age 2 weeks and greatly increased at age 8 weeks in TG T400N mice relative to WT littermates, whereas there was no apparent difference in GLUT4 expression ( n = 10 − 12 / group, pooled into one lane). The α1 subunit of the Na + /K + -ATPase, a sarcolemmal membrane marker, was used to document adequate enrichment of membrane fractions. (E)Immunofluorescence microscopy showed that SGLT1 (green) had similar subcellular distributions in WT and TG T400N cardiac myocytes at ages 2 and 8 weeks. (F) Immunofluorescence microscopy showed that α-subunit Na + /K + -ATPase (green, upper panel) and SGLT1 (red, middle panel) partially co-localized (lower panel) with in cardiac myocytes from 8 week old WT and TG T400N mice. (G) Control incubations were performed with secondary antibody only (“no-primary” control) and with nonspecific primary antibody, and showed no immunofluorescence labeling under identical acquisition conditions. Open bars, WT; closed bars, TG T400N . T, TG T400N ; W, WT. Molecular weights (MW) on immunoblots were estimated by protein marker sizes. Coomassie blue staining was used to document the relative quantity of protein loaded for the immunoblots and to normalize densitometry analysis. Scale bars on photomicrographs represent 37.5 μm. *, P

    Journal: Journal of molecular and cellular cardiology

    Article Title: SGLT1, a Novel Cardiac Glucose Transporter, Mediates Increased Glucose Uptake in PRKAG2 Cardiomyopathy

    doi: 10.1016/j.yjmcc.2010.06.003

    Figure Lengend Snippet: Cardiac GLUT1, GLUT4, and SGLT1 mRNA and protein expression in TG T400N mice. (A) Total cardiac GLUT1 and GLUT4 transcript expression, as assessed by QPCR, were similar in TG T400N mice and WT littermates at ages 2 and 8 weeks, whereas cardiac SGLT1 transcript expression was increased in TG T400N mice at ages 2 and 8 weeks ( n = 3 / group). (B) In whole heart homogenates, densitometry analysis of immunoblots showed that GLUT1 protein was significantly increased, GLUT4 protein was significantly decreased, and SGLT1 protein was unchanged in TG T400N mice relative to WT littermates at ages 2 and 8 weeks. (C) In total membrane extracts, densitometry analysis of immunoblots showed that GLUT1 and GLUT4 protein expression was not significantly different in TG T400N mice relative to WT littermates at ages 2 and 8 weeks, whereas SGLT1 protein expression was significantly increased in TG T400N mice at ages 2 and 8 weeks. (D) In sarcolemmal membrane extracts, GLUT1 and SGLT1 protein expression appeared to be mildly increased at age 2 weeks and greatly increased at age 8 weeks in TG T400N mice relative to WT littermates, whereas there was no apparent difference in GLUT4 expression ( n = 10 − 12 / group, pooled into one lane). The α1 subunit of the Na + /K + -ATPase, a sarcolemmal membrane marker, was used to document adequate enrichment of membrane fractions. (E)Immunofluorescence microscopy showed that SGLT1 (green) had similar subcellular distributions in WT and TG T400N cardiac myocytes at ages 2 and 8 weeks. (F) Immunofluorescence microscopy showed that α-subunit Na + /K + -ATPase (green, upper panel) and SGLT1 (red, middle panel) partially co-localized (lower panel) with in cardiac myocytes from 8 week old WT and TG T400N mice. (G) Control incubations were performed with secondary antibody only (“no-primary” control) and with nonspecific primary antibody, and showed no immunofluorescence labeling under identical acquisition conditions. Open bars, WT; closed bars, TG T400N . T, TG T400N ; W, WT. Molecular weights (MW) on immunoblots were estimated by protein marker sizes. Coomassie blue staining was used to document the relative quantity of protein loaded for the immunoblots and to normalize densitometry analysis. Scale bars on photomicrographs represent 37.5 μm. *, P

    Article Snippet: Thus, there may be differences between acute and chronic AMPK regulation of SGLT1, and differences among tissues, cell types, and possibly species.

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Western Blot, Marker, Microscopy, Immunofluorescence, Labeling, Staining

    Increased cardiac glucose uptake and glycogen deposition in TG T400N mice was sensitive to phlorizin, a specific SGLT1 inhibitor. (A) There was no significant change in cardiac glucose uptake in 6–8 week old WT mice 10 min following acute administration of phlorizin (400 mg/kg IP). (B) In contrast, cardiac glucose uptake was reduced in 6–8 week old TG T400N mice 10 min following acute administration of phlorizin (400 mg/kg IP). (C) Chronic administration phlorizin (100 mg/kg/day) using a subcutaneous osmotic minipump in TG T400N mice from age 2 weeks through age 6 weeks resulted in a reduction in cardiac glycogen content. Open bars, vehicle treated control mice; closed bars, phlorizin treated mice. n = 4 / group, *, P

    Journal: Journal of molecular and cellular cardiology

    Article Title: SGLT1, a Novel Cardiac Glucose Transporter, Mediates Increased Glucose Uptake in PRKAG2 Cardiomyopathy

    doi: 10.1016/j.yjmcc.2010.06.003

    Figure Lengend Snippet: Increased cardiac glucose uptake and glycogen deposition in TG T400N mice was sensitive to phlorizin, a specific SGLT1 inhibitor. (A) There was no significant change in cardiac glucose uptake in 6–8 week old WT mice 10 min following acute administration of phlorizin (400 mg/kg IP). (B) In contrast, cardiac glucose uptake was reduced in 6–8 week old TG T400N mice 10 min following acute administration of phlorizin (400 mg/kg IP). (C) Chronic administration phlorizin (100 mg/kg/day) using a subcutaneous osmotic minipump in TG T400N mice from age 2 weeks through age 6 weeks resulted in a reduction in cardiac glycogen content. Open bars, vehicle treated control mice; closed bars, phlorizin treated mice. n = 4 / group, *, P

    Article Snippet: Thus, there may be differences between acute and chronic AMPK regulation of SGLT1, and differences among tissues, cell types, and possibly species.

    Techniques: Mouse Assay

    A hypothetical model of the mechanism by which increased AMPK activity leads to increased cardiac glucose uptake via SGLT1 in PRKAG2 cardiomyopathy. AMPK directly or indirectly phosphorylates the transcription factor Sp1, which increases binding of Sp1 to the promoter of the SLC5A1 gene encoding SGLT. Binding of the transcription factor HNF-1 is also increased by unknown mechanisms. These factors increase transcription of SLC5A1 . AMPK stimulates binding of the protein HuR to a critical uridine-rich element (URE) in the 3’ untranslated region of the SGLT1 transcript, increasing its stability. AMPK directly or indirectly phosphorylates the translated SGLT1 protein, increasing translocation of SGLT1 to the sarcolemma. Increased sarcolemmal SGLT1 then leads to increased cardiac glucose uptake.

    Journal: Journal of molecular and cellular cardiology

    Article Title: SGLT1, a Novel Cardiac Glucose Transporter, Mediates Increased Glucose Uptake in PRKAG2 Cardiomyopathy

    doi: 10.1016/j.yjmcc.2010.06.003

    Figure Lengend Snippet: A hypothetical model of the mechanism by which increased AMPK activity leads to increased cardiac glucose uptake via SGLT1 in PRKAG2 cardiomyopathy. AMPK directly or indirectly phosphorylates the transcription factor Sp1, which increases binding of Sp1 to the promoter of the SLC5A1 gene encoding SGLT. Binding of the transcription factor HNF-1 is also increased by unknown mechanisms. These factors increase transcription of SLC5A1 . AMPK stimulates binding of the protein HuR to a critical uridine-rich element (URE) in the 3’ untranslated region of the SGLT1 transcript, increasing its stability. AMPK directly or indirectly phosphorylates the translated SGLT1 protein, increasing translocation of SGLT1 to the sarcolemma. Increased sarcolemmal SGLT1 then leads to increased cardiac glucose uptake.

    Article Snippet: Thus, there may be differences between acute and chronic AMPK regulation of SGLT1, and differences among tissues, cell types, and possibly species.

    Techniques: Activity Assay, Binding Assay, Translocation Assay

    Reduced ALK1 Expression Does Not Affect Cardiac Hypertrophy or Myocardial Capillary Density A and B, Representative histological staining and quantification of LV cardiomyocyte cross sectional area in WT and Alk1 +/− mice after two weeks of TAC (n=6/group). C to E, LV mRNA levels of beta-myosin heavy chain (MHC), sarcoplasmic endoplasmic reticulum ATPase (SERCA) and calcineurin. F and G, Representative immunostaining and quantification of myocardial capillary density. p

    Journal: Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology

    Article Title: Reduced Activin Receptor Like-Kinase 1 Activity Promotes Cardiac Fibrosis in Heart Failure

    doi: 10.1016/j.carpath.2017.07.004

    Figure Lengend Snippet: Reduced ALK1 Expression Does Not Affect Cardiac Hypertrophy or Myocardial Capillary Density A and B, Representative histological staining and quantification of LV cardiomyocyte cross sectional area in WT and Alk1 +/− mice after two weeks of TAC (n=6/group). C to E, LV mRNA levels of beta-myosin heavy chain (MHC), sarcoplasmic endoplasmic reticulum ATPase (SERCA) and calcineurin. F and G, Representative immunostaining and quantification of myocardial capillary density. p

    Article Snippet: The mechanism by which ALK1 regulates cardiac fibrosis in HF remains poorly understood.

    Techniques: Expressing, Staining, Mouse Assay, Immunostaining

    Smad 1 Phosphorylation and Activity is Attenuated in Alk1 +/− Mice After Severe Pressure Overload Induced Heart Failure A, Representative Western blot of phosphorylated SMAD3 (pSmad3), total SMAD3 and GAPDH. Quantification of pSMAD3 to total SMAD3 is shown in inset. B, Representative Western blot of phosphorylated SMAD1 (pSmad1), total SMAD1 and GAPDH. For pSMAD1 levels, the antibody used detects pSMAD1/5/8. The topmost band at 60 kD representing pSmad 1 was used for quantification of pSMAD1 levels. Quantification of pSMAD1 to total SMAD1 is shown in inset. C and D, LV mRNA levels of Id1 and Id2. p

    Journal: Cardiovascular pathology : the official journal of the Society for Cardiovascular Pathology

    Article Title: Reduced Activin Receptor Like-Kinase 1 Activity Promotes Cardiac Fibrosis in Heart Failure

    doi: 10.1016/j.carpath.2017.07.004

    Figure Lengend Snippet: Smad 1 Phosphorylation and Activity is Attenuated in Alk1 +/− Mice After Severe Pressure Overload Induced Heart Failure A, Representative Western blot of phosphorylated SMAD3 (pSmad3), total SMAD3 and GAPDH. Quantification of pSMAD3 to total SMAD3 is shown in inset. B, Representative Western blot of phosphorylated SMAD1 (pSmad1), total SMAD1 and GAPDH. For pSMAD1 levels, the antibody used detects pSMAD1/5/8. The topmost band at 60 kD representing pSmad 1 was used for quantification of pSMAD1 levels. Quantification of pSMAD1 to total SMAD1 is shown in inset. C and D, LV mRNA levels of Id1 and Id2. p

    Article Snippet: The mechanism by which ALK1 regulates cardiac fibrosis in HF remains poorly understood.

    Techniques: Activity Assay, Mouse Assay, Western Blot

    O -GlcNAc modification is essential for cellular survival and Bcl-2 translocation. A) Representative immunoblotting CTD110.6 and OGT and mean intensity of all O -GlcNAc proteins and OGT of the total whole cell lysate determined by densitometric analysis.

    Journal: American journal of physiology. Cell physiology

    Article Title: Glucosamine Protects Neonatal Cardiomyocytes from Ischemia-Reperfusion Injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

    doi: 10.1152/ajpcell.00456.2007

    Figure Lengend Snippet: O -GlcNAc modification is essential for cellular survival and Bcl-2 translocation. A) Representative immunoblotting CTD110.6 and OGT and mean intensity of all O -GlcNAc proteins and OGT of the total whole cell lysate determined by densitometric analysis.

    Article Snippet: Bcl-2 has been shown to block p53-mediated apoptosis in cardiac myocytes ( ) and overexpression of Bcl-2 suppressed both p53-dependent and p53-independent activation of the intrinsic death pathway ( ).

    Techniques: Modification, Translocation Assay

    Glucosamine had no effect on Bcl-2 family proteins, Bcl-2, Bax, and Bad, but enhanced mitochondrial Bcl-2 translocation. A) Representative Bcl-2, Bax, and Bad immunoblots of the whole cell lysate of NRVMs. Equal protein loading was confirmed by β-actin

    Journal: American journal of physiology. Cell physiology

    Article Title: Glucosamine Protects Neonatal Cardiomyocytes from Ischemia-Reperfusion Injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

    doi: 10.1152/ajpcell.00456.2007

    Figure Lengend Snippet: Glucosamine had no effect on Bcl-2 family proteins, Bcl-2, Bax, and Bad, but enhanced mitochondrial Bcl-2 translocation. A) Representative Bcl-2, Bax, and Bad immunoblots of the whole cell lysate of NRVMs. Equal protein loading was confirmed by β-actin

    Article Snippet: Bcl-2 has been shown to block p53-mediated apoptosis in cardiac myocytes ( ) and overexpression of Bcl-2 suppressed both p53-dependent and p53-independent activation of the intrinsic death pathway ( ).

    Techniques: Translocation Assay, Western Blot

    Increasing O -GlcNAc transferase (OGT) and blocking O -GlcNAcase by NButGT had different effects on O -GlcNAc protein modification, cellular survival and Bcl-2 translocation. A) Representative immunoblotting CTD110.6 and OGT and mean intensity of all O -GlcNAc

    Journal: American journal of physiology. Cell physiology

    Article Title: Glucosamine Protects Neonatal Cardiomyocytes from Ischemia-Reperfusion Injury via increased protein O-GlcNAc and increased mitochondrial Bcl-2

    doi: 10.1152/ajpcell.00456.2007

    Figure Lengend Snippet: Increasing O -GlcNAc transferase (OGT) and blocking O -GlcNAcase by NButGT had different effects on O -GlcNAc protein modification, cellular survival and Bcl-2 translocation. A) Representative immunoblotting CTD110.6 and OGT and mean intensity of all O -GlcNAc

    Article Snippet: Bcl-2 has been shown to block p53-mediated apoptosis in cardiac myocytes ( ) and overexpression of Bcl-2 suppressed both p53-dependent and p53-independent activation of the intrinsic death pathway ( ).

    Techniques: Blocking Assay, Modification, Translocation Assay

    (a) Endothelin-1 (ET-1) gene expression in MPS bioreactor constructs vs. constructs engineered in plate. (b) and (c) representative IHC pictures of PCL and PCL/HA based constructs respectively, stained with anti-pAKT antibody: positive red signals are pointed by arrows. Scale bar = 100 μm

    Journal: bioRxiv

    Article Title: An in vitro Chondro-osteo-vascular Triphasic Model of the Osteochondral Complex

    doi: 10.1101/2020.08.27.270660

    Figure Lengend Snippet: (a) Endothelin-1 (ET-1) gene expression in MPS bioreactor constructs vs. constructs engineered in plate. (b) and (c) representative IHC pictures of PCL and PCL/HA based constructs respectively, stained with anti-pAKT antibody: positive red signals are pointed by arrows. Scale bar = 100 μm

    Article Snippet: Human osteopontin (OPN), osteocalcin (OSC) and bone sialoprotein 2 (BSP2) were analyzed to assess osteogenic differentiation; aggrecan (ACAN), SRY (sex determining region Y)-box 9 (SOX9) and collagen type II (COL2) were analyzed to assess chondrogenic differentiation; and endothelin-1 (ET-1) was analyzed for HUVECs activity. hMSCs or hMSCs/HUVECs seeded in the scaffolds and cultured in GM after 10 days of proliferation were used as “day 0” for osteogenesis; cells resuspended in the gelMA solution prior to differentiation were used as “day 0” for chondrogenesis.

    Techniques: Expressing, Construct, Immunohistochemistry, Staining

    Altered exosomal contents by overexpression of Hsp20 in cardiomyocytes could be delivered to endothelial cells and native cardiomyocytes. Representative Western blots ( A ) and their quantitative results ( B ) showed that higher levels of Hsp20, survivin, and p-Akt encased in TG-Exo than WT-Exo. C : SOD1 levels were increased in TG-Exo, compared with WT-Exo ( n = 4 for A – C ). * P

    Journal: Diabetes

    Article Title: Hsp20-Mediated Activation of Exosome Biogenesis in Cardiomyocytes Improves Cardiac Function and Angiogenesis in Diabetic Mice

    doi: 10.2337/db15-1563

    Figure Lengend Snippet: Altered exosomal contents by overexpression of Hsp20 in cardiomyocytes could be delivered to endothelial cells and native cardiomyocytes. Representative Western blots ( A ) and their quantitative results ( B ) showed that higher levels of Hsp20, survivin, and p-Akt encased in TG-Exo than WT-Exo. C : SOD1 levels were increased in TG-Exo, compared with WT-Exo ( n = 4 for A – C ). * P

    Article Snippet: Thus, this study for the first time shows that cardiac-specific overexpression of Hsp20 remarkably attenuated diabetes-induced cardiac dysfunction and adverse remodeling.

    Techniques: Over Expression, Western Blot

    Injection of Hsp20-enriched exosomes collected from TG cardiomyocytes protects mice against STZ-induced cardiac adverse remodeling. A : DiR-labeled cardiomyocyte-derived exosomes were detectable in mouse cardiomyocytes 1 h after the tail-vein injection in vivo. The right image is magnified from insert square of A . B and C : Exosomal Hsp20 was dose-dependently delivered to the mouse heart after the tail-vein injection. D and E : The time course determination of cardiac Hsp20 levels in TG-Exo–injected mice. F : A scheme of the experimental procedure for the exosome injection in STZ-treated mice. G and H : LVIDd and LVEF% were significantly improved in TG-Exo–injected diabetic mice ( n = 5–8). * P

    Journal: Diabetes

    Article Title: Hsp20-Mediated Activation of Exosome Biogenesis in Cardiomyocytes Improves Cardiac Function and Angiogenesis in Diabetic Mice

    doi: 10.2337/db15-1563

    Figure Lengend Snippet: Injection of Hsp20-enriched exosomes collected from TG cardiomyocytes protects mice against STZ-induced cardiac adverse remodeling. A : DiR-labeled cardiomyocyte-derived exosomes were detectable in mouse cardiomyocytes 1 h after the tail-vein injection in vivo. The right image is magnified from insert square of A . B and C : Exosomal Hsp20 was dose-dependently delivered to the mouse heart after the tail-vein injection. D and E : The time course determination of cardiac Hsp20 levels in TG-Exo–injected mice. F : A scheme of the experimental procedure for the exosome injection in STZ-treated mice. G and H : LVIDd and LVEF% were significantly improved in TG-Exo–injected diabetic mice ( n = 5–8). * P

    Article Snippet: Thus, this study for the first time shows that cardiac-specific overexpression of Hsp20 remarkably attenuated diabetes-induced cardiac dysfunction and adverse remodeling.

    Techniques: Injection, Mouse Assay, Labeling, Derivative Assay, In Vivo

    Hsp20-mediated cardioprotective effects are largely offset by blockade of the exosome generation. A : A scheme of the experimental procedure for the treatment of mice with STZ and GW4869. B : Exosome concentration was measured in the serum of mice treated with GW4869 ( n = 5). * P

    Journal: Diabetes

    Article Title: Hsp20-Mediated Activation of Exosome Biogenesis in Cardiomyocytes Improves Cardiac Function and Angiogenesis in Diabetic Mice

    doi: 10.2337/db15-1563

    Figure Lengend Snippet: Hsp20-mediated cardioprotective effects are largely offset by blockade of the exosome generation. A : A scheme of the experimental procedure for the treatment of mice with STZ and GW4869. B : Exosome concentration was measured in the serum of mice treated with GW4869 ( n = 5). * P

    Article Snippet: Thus, this study for the first time shows that cardiac-specific overexpression of Hsp20 remarkably attenuated diabetes-induced cardiac dysfunction and adverse remodeling.

    Techniques: Mouse Assay, Concentration Assay

    Cardiac-specific overexpression of Hsp20 attenuates STZ-induced cardiac dysfunction and remodeling. A and B : Western blotting results showed that Hsp20 was overexpressed by 10-fold in TG mouse hearts. α-Actin was used as a loading control ( n = 4). * P

    Journal: Diabetes

    Article Title: Hsp20-Mediated Activation of Exosome Biogenesis in Cardiomyocytes Improves Cardiac Function and Angiogenesis in Diabetic Mice

    doi: 10.2337/db15-1563

    Figure Lengend Snippet: Cardiac-specific overexpression of Hsp20 attenuates STZ-induced cardiac dysfunction and remodeling. A and B : Western blotting results showed that Hsp20 was overexpressed by 10-fold in TG mouse hearts. α-Actin was used as a loading control ( n = 4). * P

    Article Snippet: Thus, this study for the first time shows that cardiac-specific overexpression of Hsp20 remarkably attenuated diabetes-induced cardiac dysfunction and adverse remodeling.

    Techniques: Over Expression, Western Blot

    Hsp20 promotes exosome biogenesis via interacting with Tsg101. A : Diagram of exosome biogenesis/release pathway. B : Overexpression of Hsp20 in the heart increased the expression levels of major mediators involved in exosome generation ( n = 3). * P

    Journal: Diabetes

    Article Title: Hsp20-Mediated Activation of Exosome Biogenesis in Cardiomyocytes Improves Cardiac Function and Angiogenesis in Diabetic Mice

    doi: 10.2337/db15-1563

    Figure Lengend Snippet: Hsp20 promotes exosome biogenesis via interacting with Tsg101. A : Diagram of exosome biogenesis/release pathway. B : Overexpression of Hsp20 in the heart increased the expression levels of major mediators involved in exosome generation ( n = 3). * P

    Article Snippet: Thus, this study for the first time shows that cardiac-specific overexpression of Hsp20 remarkably attenuated diabetes-induced cardiac dysfunction and adverse remodeling.

    Techniques: Over Expression, Expressing

    Exosomes derived from Hsp20-overexpressing cardiomyocytes protect myocardial endothelial cells and cardiomyocytes against HG-induced stress conditions. The size of exosomes derived from WT cardiomyocytes ( A ) and Hsp20-TG cardiomyocytes ( B ), measured using a Zetasizer Nano ZS instrument. C : Protein levels of CD63 and CD81, two exosome markers, were similarly encased in WT-Exo and TG-Exo. D : Effects of WT-Exo (collected from the supernatants of WT cardiomyocytes) and TG-Exo (collected from the supernatants of TG cardiomyocytes) on HG-induced endothelial cell growth ( n = 6 wells). * P

    Journal: Diabetes

    Article Title: Hsp20-Mediated Activation of Exosome Biogenesis in Cardiomyocytes Improves Cardiac Function and Angiogenesis in Diabetic Mice

    doi: 10.2337/db15-1563

    Figure Lengend Snippet: Exosomes derived from Hsp20-overexpressing cardiomyocytes protect myocardial endothelial cells and cardiomyocytes against HG-induced stress conditions. The size of exosomes derived from WT cardiomyocytes ( A ) and Hsp20-TG cardiomyocytes ( B ), measured using a Zetasizer Nano ZS instrument. C : Protein levels of CD63 and CD81, two exosome markers, were similarly encased in WT-Exo and TG-Exo. D : Effects of WT-Exo (collected from the supernatants of WT cardiomyocytes) and TG-Exo (collected from the supernatants of TG cardiomyocytes) on HG-induced endothelial cell growth ( n = 6 wells). * P

    Article Snippet: Thus, this study for the first time shows that cardiac-specific overexpression of Hsp20 remarkably attenuated diabetes-induced cardiac dysfunction and adverse remodeling.

    Techniques: Derivative Assay

    Hsp20 exosomes suppress HG-induced oxidative stress in endothelial cells and cardiomyocytes. The amount of SOD1 was increased in exosome-treated endothelial cells ( A ) and cardiomyocytes ( B ) under normal conditions. HG-induced decrease in the amount of SOD1 was improved to a greater degree in TG-Exo–treated cells than WT-Exo–treated samples ( A and B ). By contrast, HG-triggered increase of ROS levels was attenuated to a greater degree in TG-Exo–treated endothelial cells ( C )/cardiomyocytes ( D ) than WT-Exo–treated samples ( C and D ) ( n = 3 wells). * P

    Journal: Diabetes

    Article Title: Hsp20-Mediated Activation of Exosome Biogenesis in Cardiomyocytes Improves Cardiac Function and Angiogenesis in Diabetic Mice

    doi: 10.2337/db15-1563

    Figure Lengend Snippet: Hsp20 exosomes suppress HG-induced oxidative stress in endothelial cells and cardiomyocytes. The amount of SOD1 was increased in exosome-treated endothelial cells ( A ) and cardiomyocytes ( B ) under normal conditions. HG-induced decrease in the amount of SOD1 was improved to a greater degree in TG-Exo–treated cells than WT-Exo–treated samples ( A and B ). By contrast, HG-triggered increase of ROS levels was attenuated to a greater degree in TG-Exo–treated endothelial cells ( C )/cardiomyocytes ( D ) than WT-Exo–treated samples ( C and D ) ( n = 3 wells). * P

    Article Snippet: Thus, this study for the first time shows that cardiac-specific overexpression of Hsp20 remarkably attenuated diabetes-induced cardiac dysfunction and adverse remodeling.

    Techniques:

    Arrb2/miR-155/GSK3β pathway is important in CSC-mediated cardiac repair. Isolated 2 × 10 5 Sca-1+ cells from WT or Arrb2-KO mice were injected immediately into infarcted and border zones of the mouse heart after myocardial infarction (MI). Hearts were then reperfused for 1 hr. After 2 weeks, 2-mm sections of hearts near the mid-ventricles were collected. ( A ) Fluoresence microscopy of hearts for WT mice with MI injected with WT Sca-1+ cells and stained with cTnT. Red shows cardiomyocytes; green shows injected Sca-1+ cells; blue shows DAPI-stained cell nuclei; scale bar, 40 μm; n = 6. ( B ) Western blot analysis of the expression of Arrb2, total Akt and p-Akt, and total and p-GSK3β. GADPH was a loading control. The column shows the quantification of the protein expression. Protein levels were normalized to GAPDH or total protein; n = 3; * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: β-arrestin2/miR-155/GSK3β regulates transition of 5′-azacytizine-induced Sca-1-positive cells to cardiomyocytes

    doi: 10.1111/jcmm.12339

    Figure Lengend Snippet: Arrb2/miR-155/GSK3β pathway is important in CSC-mediated cardiac repair. Isolated 2 × 10 5 Sca-1+ cells from WT or Arrb2-KO mice were injected immediately into infarcted and border zones of the mouse heart after myocardial infarction (MI). Hearts were then reperfused for 1 hr. After 2 weeks, 2-mm sections of hearts near the mid-ventricles were collected. ( A ) Fluoresence microscopy of hearts for WT mice with MI injected with WT Sca-1+ cells and stained with cTnT. Red shows cardiomyocytes; green shows injected Sca-1+ cells; blue shows DAPI-stained cell nuclei; scale bar, 40 μm; n = 6. ( B ) Western blot analysis of the expression of Arrb2, total Akt and p-Akt, and total and p-GSK3β. GADPH was a loading control. The column shows the quantification of the protein expression. Protein levels were normalized to GAPDH or total protein; n = 3; * P

    Article Snippet: We used the 5aza-induced differentiation model in vitro , and showed that Arrb2 could promote the differentiation of Sca-1+ cells to cardiomyocytes, which suggested an important role of Arrb2 in Sca-1+ cell transition and promoted us to explore the mechanisms of Arrb2 mediated Sca-1+ CSCs transition to cardiomyocyte.

    Techniques: Isolation, Mouse Assay, Injection, Microscopy, Staining, Western Blot, Expressing

    GSK3β is involved in 5aza-induced differentiation of CSCs to cardiomyocytes. ( A ) Sca-1+ cells from WT mice were treated with 5aza as in Figure 1 A and incubated with or without SB216763 at 10 μM for the first 3 days. The mRNA expression of MYH6 and cTnT was analysed by RT-PCR analysis. ( B ) Sca-1+ cells from WT and Arrb2-KO mice were treated with 5aza as in Figure 1 A. The expression of total and phosphorylated Akt (p-Akt), total and p-GSK3β were analysed by western blot. ( C ) Quantification of p-Akt levels shown in B . protein level were normalized to AKT. ( D ) Quantification of p-GSK3β levels shown in B . protein level were normalized to GSK3β. Data are mean ± SEM of four experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: β-arrestin2/miR-155/GSK3β regulates transition of 5′-azacytizine-induced Sca-1-positive cells to cardiomyocytes

    doi: 10.1111/jcmm.12339

    Figure Lengend Snippet: GSK3β is involved in 5aza-induced differentiation of CSCs to cardiomyocytes. ( A ) Sca-1+ cells from WT mice were treated with 5aza as in Figure 1 A and incubated with or without SB216763 at 10 μM for the first 3 days. The mRNA expression of MYH6 and cTnT was analysed by RT-PCR analysis. ( B ) Sca-1+ cells from WT and Arrb2-KO mice were treated with 5aza as in Figure 1 A. The expression of total and phosphorylated Akt (p-Akt), total and p-GSK3β were analysed by western blot. ( C ) Quantification of p-Akt levels shown in B . protein level were normalized to AKT. ( D ) Quantification of p-GSK3β levels shown in B . protein level were normalized to GSK3β. Data are mean ± SEM of four experiments. * P

    Article Snippet: We used the 5aza-induced differentiation model in vitro , and showed that Arrb2 could promote the differentiation of Sca-1+ cells to cardiomyocytes, which suggested an important role of Arrb2 in Sca-1+ cell transition and promoted us to explore the mechanisms of Arrb2 mediated Sca-1+ CSCs transition to cardiomyocyte.

    Techniques: Mouse Assay, Incubation, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Arrb2 is a miR-155 target. ( A ) Sequence alignment of miR-155 and its target site in the 3′-UTR of Arrb2 (downloaded from http://www.targetscan.org ). ( B ) The seed region of Arrb2 3′-UTR was mutated as indicated. ( C ) HEK293T cells were cotransfected with 60 ng miR-155 plasmid or empty EGFP plasmid control and 0.1 μg psicheck2 3′-UTR-WT (WT Arrb2) or psicheck2 3′-UTR-MUT (mutant miR-155 target site in Arrb2 3′-UTR). Cells were collected 48 hrs after transfection and analysed by dual luciferase reporter assay. The psicheck2 vector that provided the constitutive expression of Renilla luciferase was cotransfected as an internal control. Data are mean ± SEM of four experiments. ** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: β-arrestin2/miR-155/GSK3β regulates transition of 5′-azacytizine-induced Sca-1-positive cells to cardiomyocytes

    doi: 10.1111/jcmm.12339

    Figure Lengend Snippet: Arrb2 is a miR-155 target. ( A ) Sequence alignment of miR-155 and its target site in the 3′-UTR of Arrb2 (downloaded from http://www.targetscan.org ). ( B ) The seed region of Arrb2 3′-UTR was mutated as indicated. ( C ) HEK293T cells were cotransfected with 60 ng miR-155 plasmid or empty EGFP plasmid control and 0.1 μg psicheck2 3′-UTR-WT (WT Arrb2) or psicheck2 3′-UTR-MUT (mutant miR-155 target site in Arrb2 3′-UTR). Cells were collected 48 hrs after transfection and analysed by dual luciferase reporter assay. The psicheck2 vector that provided the constitutive expression of Renilla luciferase was cotransfected as an internal control. Data are mean ± SEM of four experiments. ** P

    Article Snippet: We used the 5aza-induced differentiation model in vitro , and showed that Arrb2 could promote the differentiation of Sca-1+ cells to cardiomyocytes, which suggested an important role of Arrb2 in Sca-1+ cell transition and promoted us to explore the mechanisms of Arrb2 mediated Sca-1+ CSCs transition to cardiomyocyte.

    Techniques: Sequencing, Plasmid Preparation, Mutagenesis, Transfection, Luciferase, Reporter Assay, Expressing

    Effect of Arrb2 on 5′-azacytizine-induced differentiation of cardiac stem cells (CSCs) to cardiomyocytes. ( A ) Isolated Sca-1+ cells from wild-type (WT) mice were seeded 1 day before cells were treated with 5′-azacytizine (5aza) at 10 μM. After 3 days' treatment, cell culture medium was changed every 3 days for 2 and 3 weeks. Relative gene expression of cardiomyocyte markers including MYH6, GATA4, and cTnT were detected by RT-PCR. ( B and C ) isolated Sca-1+ cells from wild-type (WT) mice were treated with 5aza at 10 μM for 3 weeks. Arrb2 expression was determined by RT-PCR ( B ) and western blot analysis ( C ). ( D and E ) Sca-1+ cells from WT mice were transfected with full-length Arrb2 or control vector. After 24 hrs, cells were treated with 5aza as in A ; the level of cTnT was detected by fluorescence assay ( D ) and the expression of MYH6, GATA4 and cTnT by RT-PCR ( E ). ( D ) It shows phase-contrast (transmission) and fluorescence images. GFP shows transfected cells; scale bar = 15 μm. ( F ) Sca-1+ cells from WT and Arrb2-knockout (KO) mice were treated with 5aza as in A . Real-time PCR analysis of the mRNA levels of MYH6, GATA4, and cTnT. Data are mean ± SEM of three experiments. * P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: β-arrestin2/miR-155/GSK3β regulates transition of 5′-azacytizine-induced Sca-1-positive cells to cardiomyocytes

    doi: 10.1111/jcmm.12339

    Figure Lengend Snippet: Effect of Arrb2 on 5′-azacytizine-induced differentiation of cardiac stem cells (CSCs) to cardiomyocytes. ( A ) Isolated Sca-1+ cells from wild-type (WT) mice were seeded 1 day before cells were treated with 5′-azacytizine (5aza) at 10 μM. After 3 days' treatment, cell culture medium was changed every 3 days for 2 and 3 weeks. Relative gene expression of cardiomyocyte markers including MYH6, GATA4, and cTnT were detected by RT-PCR. ( B and C ) isolated Sca-1+ cells from wild-type (WT) mice were treated with 5aza at 10 μM for 3 weeks. Arrb2 expression was determined by RT-PCR ( B ) and western blot analysis ( C ). ( D and E ) Sca-1+ cells from WT mice were transfected with full-length Arrb2 or control vector. After 24 hrs, cells were treated with 5aza as in A ; the level of cTnT was detected by fluorescence assay ( D ) and the expression of MYH6, GATA4 and cTnT by RT-PCR ( E ). ( D ) It shows phase-contrast (transmission) and fluorescence images. GFP shows transfected cells; scale bar = 15 μm. ( F ) Sca-1+ cells from WT and Arrb2-knockout (KO) mice were treated with 5aza as in A . Real-time PCR analysis of the mRNA levels of MYH6, GATA4, and cTnT. Data are mean ± SEM of three experiments. * P

    Article Snippet: We used the 5aza-induced differentiation model in vitro , and showed that Arrb2 could promote the differentiation of Sca-1+ cells to cardiomyocytes, which suggested an important role of Arrb2 in Sca-1+ cell transition and promoted us to explore the mechanisms of Arrb2 mediated Sca-1+ CSCs transition to cardiomyocyte.

    Techniques: Isolation, Mouse Assay, Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Plasmid Preparation, Fluorescence, Transmission Assay, Knock-Out, Real-time Polymerase Chain Reaction

    MiR-155 inhibits 5aza-induced differentiation of CSCs into cardiomyocytes through Arrb2. ( A ) RT-PCR analysis of the expression of miR-155 in Sca-1+ cells from WT mice treated as in Figure 1 A. ( B ) Sca-1+ cells from WT mice were transfected with miR-155 plasmid or empty plasmid control. After 24 hrs, cells were treated with 5aza and the expression of MYH6 and cTnT was examined by RT-PCR. ( C ) Sca-1+ cells from WT and Arrb2-KO mice were treated with 5aza and miR-155 expression was examined as in A . Data are mean ± SEM of three experiments. ** P

    Journal: Journal of Cellular and Molecular Medicine

    Article Title: β-arrestin2/miR-155/GSK3β regulates transition of 5′-azacytizine-induced Sca-1-positive cells to cardiomyocytes

    doi: 10.1111/jcmm.12339

    Figure Lengend Snippet: MiR-155 inhibits 5aza-induced differentiation of CSCs into cardiomyocytes through Arrb2. ( A ) RT-PCR analysis of the expression of miR-155 in Sca-1+ cells from WT mice treated as in Figure 1 A. ( B ) Sca-1+ cells from WT mice were transfected with miR-155 plasmid or empty plasmid control. After 24 hrs, cells were treated with 5aza and the expression of MYH6 and cTnT was examined by RT-PCR. ( C ) Sca-1+ cells from WT and Arrb2-KO mice were treated with 5aza and miR-155 expression was examined as in A . Data are mean ± SEM of three experiments. ** P

    Article Snippet: We used the 5aza-induced differentiation model in vitro , and showed that Arrb2 could promote the differentiation of Sca-1+ cells to cardiomyocytes, which suggested an important role of Arrb2 in Sca-1+ cell transition and promoted us to explore the mechanisms of Arrb2 mediated Sca-1+ CSCs transition to cardiomyocyte.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Mouse Assay, Transfection, Plasmid Preparation

    Confocal photomicrographs demonstrating internalized PNP (120 nm amidine-modified) and clathrin heavy chain in MDCK-II after 1 h of apical exposure to 176 μg/mL PNP in the absence of dansylcadaverine (Panel A). PNP appear in green, nuclei in blue and clathrin heavy chain in red. PNP colocalizes with clathrin heavy chain, appearing yellowish-green. Panel B illustrates the colocalization profile of PNP (green) and clathrin heavy chain (red), where color intensities from green and red channels are estimated along the line segment shown in panel A. As can be appreciated, green intensity peaks coincide with red intensity peaks. The numbers 1–5 in panel B correspond to those in panel A, respectively. Panel C represents a typical confocal image obtained from MDCK-II co-incubated apically with 200 μM dansylcadaverine and 120 nm amidine-modified PNP for 1 h. Comparatively few PNP (green) can be seen in Panel C. No colocalization of PNP and clathrin heavy chain (red) is detectable in Panel C.

    Journal: Nanomedicine : nanotechnology, biology, and medicine

    Article Title: Polystyrene nanoparticle trafficking across MDCK-II

    doi: 10.1016/j.nano.2011.01.008

    Figure Lengend Snippet: Confocal photomicrographs demonstrating internalized PNP (120 nm amidine-modified) and clathrin heavy chain in MDCK-II after 1 h of apical exposure to 176 μg/mL PNP in the absence of dansylcadaverine (Panel A). PNP appear in green, nuclei in blue and clathrin heavy chain in red. PNP colocalizes with clathrin heavy chain, appearing yellowish-green. Panel B illustrates the colocalization profile of PNP (green) and clathrin heavy chain (red), where color intensities from green and red channels are estimated along the line segment shown in panel A. As can be appreciated, green intensity peaks coincide with red intensity peaks. The numbers 1–5 in panel B correspond to those in panel A, respectively. Panel C represents a typical confocal image obtained from MDCK-II co-incubated apically with 200 μM dansylcadaverine and 120 nm amidine-modified PNP for 1 h. Comparatively few PNP (green) can be seen in Panel C. No colocalization of PNP and clathrin heavy chain (red) is detectable in Panel C.

    Article Snippet: Monolayers were bathed on both sides with culture medium containing 10–200 μM methyl-β-cyclodextrin (Sigma), 200 μM monodansylcadaverine (Sigma) or 80 μM dynasore (Sigma) for 30 min prior to replacing apical fluid at t = 0 with fresh culture medium containing both 176 μg/mL of various PNP and specific endocytosis inhibitors.

    Techniques: Modification, Incubation

    Effects of inhibition of lipid raft-mediated, clathrin-mediated and dynamin-dependent endocytosis on flux of PNP (amidine-modified 20 and 120 nm) at an apical concentration of 176 μg/mL across MDCK-II. Monolayers treated with 10 μM methyl-β-cyclodextrin did not exhibit decreased PNP flux (n=6). Monolayers treated with either 200 μM dansylcadaverine or 80 μM dynasore, respectively, showed markedly decrease in PNP flux (n=6–9). * = significantly different from 20 nm, amidine-modified control. # = significantly different from 120 nm, amidine-modified control.

    Journal: Nanomedicine : nanotechnology, biology, and medicine

    Article Title: Polystyrene nanoparticle trafficking across MDCK-II

    doi: 10.1016/j.nano.2011.01.008

    Figure Lengend Snippet: Effects of inhibition of lipid raft-mediated, clathrin-mediated and dynamin-dependent endocytosis on flux of PNP (amidine-modified 20 and 120 nm) at an apical concentration of 176 μg/mL across MDCK-II. Monolayers treated with 10 μM methyl-β-cyclodextrin did not exhibit decreased PNP flux (n=6). Monolayers treated with either 200 μM dansylcadaverine or 80 μM dynasore, respectively, showed markedly decrease in PNP flux (n=6–9). * = significantly different from 20 nm, amidine-modified control. # = significantly different from 120 nm, amidine-modified control.

    Article Snippet: Monolayers were bathed on both sides with culture medium containing 10–200 μM methyl-β-cyclodextrin (Sigma), 200 μM monodansylcadaverine (Sigma) or 80 μM dynasore (Sigma) for 30 min prior to replacing apical fluid at t = 0 with fresh culture medium containing both 176 μg/mL of various PNP and specific endocytosis inhibitors.

    Techniques: Inhibition, Modification, Concentration Assay