neonatal rat cardiomyocytes  (Worthington Biochemical)


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    Name:
    Neonatal Cardiomyocyte Isolation System
    Description:
    Kit for performing five separate tissue dissociations each containing up to twelve hearts Contains single use vials of purified collagenase and trypsin CMF HBSS Leibovitz L 15 media and Falcon cell strainers along with a detailed protocol The kit is use tested by Worthington to assure performance
    Catalog Number:
    LK003300
    Price:
    256
    Size:
    1 kt
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    see components
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    Structured Review

    Worthington Biochemical neonatal rat cardiomyocytes
    Molecular and biological regenerative properties of extracellular vesicle fractions of W8B2 + CSC-conditioned medium. ( A ) Particle size distribution shows extracellular vesicles with an average modal size of 201 ± 12 nm. ( B ) Electron micrograph images of extracellular vesicles shows near-spherical shape of double membraned vesicles. ( C ) DELFIA protein analysis shows the expression of CD81 in the extracellular vesicles isolated from 2 different biological samples. The effect of extracellular vesicles on survival of ( D ) neonatal rat <t>cardiomyocytes</t> and ( E ) human cardiac microvascular endothelial cells in culture after simulated ischaemia. The effect of extracellular vesicles on ( F ) proliferation of neonatal rat cardiomyocytes proliferation and ( G ) pro-angiogenic tube formation of human cardiac microvascular endothelial cells, assessed by the number of loops formed. Data are shown as mean ± SEM from 4–6 independent experiments. *p
    Kit for performing five separate tissue dissociations each containing up to twelve hearts Contains single use vials of purified collagenase and trypsin CMF HBSS Leibovitz L 15 media and Falcon cell strainers along with a detailed protocol The kit is use tested by Worthington to assure performance
    https://www.bioz.com/result/neonatal rat cardiomyocytes/product/Worthington Biochemical
    Average 98 stars, based on 1 article reviews
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    neonatal rat cardiomyocytes - by Bioz Stars, 2021-04
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    Images

    1) Product Images from "Biologically active constituents of the secretome of human W8B2+ cardiac stem cells"

    Article Title: Biologically active constituents of the secretome of human W8B2+ cardiac stem cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19855-4

    Molecular and biological regenerative properties of extracellular vesicle fractions of W8B2 + CSC-conditioned medium. ( A ) Particle size distribution shows extracellular vesicles with an average modal size of 201 ± 12 nm. ( B ) Electron micrograph images of extracellular vesicles shows near-spherical shape of double membraned vesicles. ( C ) DELFIA protein analysis shows the expression of CD81 in the extracellular vesicles isolated from 2 different biological samples. The effect of extracellular vesicles on survival of ( D ) neonatal rat cardiomyocytes and ( E ) human cardiac microvascular endothelial cells in culture after simulated ischaemia. The effect of extracellular vesicles on ( F ) proliferation of neonatal rat cardiomyocytes proliferation and ( G ) pro-angiogenic tube formation of human cardiac microvascular endothelial cells, assessed by the number of loops formed. Data are shown as mean ± SEM from 4–6 independent experiments. *p
    Figure Legend Snippet: Molecular and biological regenerative properties of extracellular vesicle fractions of W8B2 + CSC-conditioned medium. ( A ) Particle size distribution shows extracellular vesicles with an average modal size of 201 ± 12 nm. ( B ) Electron micrograph images of extracellular vesicles shows near-spherical shape of double membraned vesicles. ( C ) DELFIA protein analysis shows the expression of CD81 in the extracellular vesicles isolated from 2 different biological samples. The effect of extracellular vesicles on survival of ( D ) neonatal rat cardiomyocytes and ( E ) human cardiac microvascular endothelial cells in culture after simulated ischaemia. The effect of extracellular vesicles on ( F ) proliferation of neonatal rat cardiomyocytes proliferation and ( G ) pro-angiogenic tube formation of human cardiac microvascular endothelial cells, assessed by the number of loops formed. Data are shown as mean ± SEM from 4–6 independent experiments. *p

    Techniques Used: Expressing, Isolation

    Biological activity profile of W8B2 + CSC-conditioned medium fractionated by ion exchange chromatography. The effect of conditioned medium on survival of ( A ) neonatal rat cardiomyocytes and ( B ) human cardiac microvascular endothelial cells. ( C ) Proliferation of neonatal rat cardiomyocytes represented by percentage of Ki67 + cells. ( D ) Pro-angiogenic tube formation of human cardiac microvascular endothelial cells, assessed by the number of loops formed in vitro . Serum-free medium and serum-free medium supplemented with 5% FCS (FCS) were served as Control and as a positive control (FCS), respectively. Data are shown as mean ± SEM from 4–6 independent experiments. *p
    Figure Legend Snippet: Biological activity profile of W8B2 + CSC-conditioned medium fractionated by ion exchange chromatography. The effect of conditioned medium on survival of ( A ) neonatal rat cardiomyocytes and ( B ) human cardiac microvascular endothelial cells. ( C ) Proliferation of neonatal rat cardiomyocytes represented by percentage of Ki67 + cells. ( D ) Pro-angiogenic tube formation of human cardiac microvascular endothelial cells, assessed by the number of loops formed in vitro . Serum-free medium and serum-free medium supplemented with 5% FCS (FCS) were served as Control and as a positive control (FCS), respectively. Data are shown as mean ± SEM from 4–6 independent experiments. *p

    Techniques Used: Activity Assay, Ion Exchange Chromatography, In Vitro, Positive Control

    2) Product Images from "BIN1 Localizes the L-Type Calcium Channel to Cardiac T-TubulesBIN1: A Protein with Great Heart"

    Article Title: BIN1 Localizes the L-Type Calcium Channel to Cardiac T-TubulesBIN1: A Protein with Great Heart

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.1000312

    BIN1 knockdown delays calcium transient development in mouse cardiomyocytes. (A) Western blot indicates an 80% knockdown of BIN1 protein by siRNA in differentiated mouse cardiomyocytes. (B) Surface biotinylation of Cav1.2 in these primary cardiomyocytes indicates a 45% reduction of surface Cav1.2 after BIN1 knockdown. (C) Live cell calcium imaging in differentiated cardiomyocytes indicates that BIN1 knockdown also delays calcium transient development in these cells. Average time to 50% maximal fluorescence intensity (T1/2 max) of calcium transient is presented in the left panel (* p
    Figure Legend Snippet: BIN1 knockdown delays calcium transient development in mouse cardiomyocytes. (A) Western blot indicates an 80% knockdown of BIN1 protein by siRNA in differentiated mouse cardiomyocytes. (B) Surface biotinylation of Cav1.2 in these primary cardiomyocytes indicates a 45% reduction of surface Cav1.2 after BIN1 knockdown. (C) Live cell calcium imaging in differentiated cardiomyocytes indicates that BIN1 knockdown also delays calcium transient development in these cells. Average time to 50% maximal fluorescence intensity (T1/2 max) of calcium transient is presented in the left panel (* p

    Techniques Used: Western Blot, Imaging, Fluorescence

    BIN1 colocalizes with Cav1.2 at T-tubules in cardiomyocytes. (A) Confocal image (100×) of human (left) and mouse (right) adult cardiomyocytes. The cells were fixed and stained with mouse anti-BIN1 or rabbit anti-Cav1.2. Two-dimensional frames of Cav1.2 and BIN1 are shown in the top panel. Cardiomyocyte fluorescence intensity profiles along the cardiomyocyte longitudinal axis are presented in the middle panel. The bottom panel is the power spectrum over spatial distance averaged from five cardiomyocytes, which indicate that both BIN1 and Cav1.2 signals occurs at every 2 µm (fundamental peak occurs at ∼2 µm). Note the small peak at 1 µm is a harmonic of the fundamental peak at 2 µm (scale bar: 10 µm). (B) Confocal images (100×) of human (left) and mouse (right) cardiomyocytes stained with mouse anti-BIN1 (green) and rabbit anti-Cav1.2 (red) reveal colocalization between BIN1 and Cav1.2 along T-tubules (scale bar: 5 µm). Pearson colocalization coefficient and scatter plot between BIN1 and Cav1.2 are also shown in this panel. (C) Electron microscopy image of adult mouse cardiomyocytes fixed and immunogold labeled for BIN1 (small dots) and Cav1.2 (large dots) (scale bar: 200 nm) (left). As seen in the enlarged image, BIN1 and Cav1.2 occurs within 50 nm on T-tubule membranes. The negative control image without primary antibodies incubation is shown at the right panel.
    Figure Legend Snippet: BIN1 colocalizes with Cav1.2 at T-tubules in cardiomyocytes. (A) Confocal image (100×) of human (left) and mouse (right) adult cardiomyocytes. The cells were fixed and stained with mouse anti-BIN1 or rabbit anti-Cav1.2. Two-dimensional frames of Cav1.2 and BIN1 are shown in the top panel. Cardiomyocyte fluorescence intensity profiles along the cardiomyocyte longitudinal axis are presented in the middle panel. The bottom panel is the power spectrum over spatial distance averaged from five cardiomyocytes, which indicate that both BIN1 and Cav1.2 signals occurs at every 2 µm (fundamental peak occurs at ∼2 µm). Note the small peak at 1 µm is a harmonic of the fundamental peak at 2 µm (scale bar: 10 µm). (B) Confocal images (100×) of human (left) and mouse (right) cardiomyocytes stained with mouse anti-BIN1 (green) and rabbit anti-Cav1.2 (red) reveal colocalization between BIN1 and Cav1.2 along T-tubules (scale bar: 5 µm). Pearson colocalization coefficient and scatter plot between BIN1 and Cav1.2 are also shown in this panel. (C) Electron microscopy image of adult mouse cardiomyocytes fixed and immunogold labeled for BIN1 (small dots) and Cav1.2 (large dots) (scale bar: 200 nm) (left). As seen in the enlarged image, BIN1 and Cav1.2 occurs within 50 nm on T-tubule membranes. The negative control image without primary antibodies incubation is shown at the right panel.

    Techniques Used: Staining, Fluorescence, Electron Microscopy, Labeling, Negative Control, Incubation

    Antegrade trafficking of Cav1.2 is microtubule dependent. (A) Surface biotinylation of adult mouse cardiomyocytes indicates that nocodazole (30 µM) progressively reduces surface Cav1.2 expression in the presence of an endocytosis inhibitor dynasore (20 µM). Note that dynasore alone significantly increases surface expression of Cav1.2 by blocking dynamin-dependent endocytosis of Cav1.2 in cardiomyocytes. (B) Top panel: Confocal images (100×) of mouse cardiomyocytes stained with rabbit anti-Cav1.2 (red) and mouse anti-α-tubulin (green) reveal localization of Cav1.2 on microtubule network (scale bar: 5 µm). Bottom panel: Deconvolution of wide-field image of HL-1 cells stained with Cav1.2 (red) and α-tubulin (green). Merged image shows localization of Cav1.2 to the microtubule network. Enlarged pictures (right) indicate that Cav1.2 is distributed along microtubules (## p
    Figure Legend Snippet: Antegrade trafficking of Cav1.2 is microtubule dependent. (A) Surface biotinylation of adult mouse cardiomyocytes indicates that nocodazole (30 µM) progressively reduces surface Cav1.2 expression in the presence of an endocytosis inhibitor dynasore (20 µM). Note that dynasore alone significantly increases surface expression of Cav1.2 by blocking dynamin-dependent endocytosis of Cav1.2 in cardiomyocytes. (B) Top panel: Confocal images (100×) of mouse cardiomyocytes stained with rabbit anti-Cav1.2 (red) and mouse anti-α-tubulin (green) reveal localization of Cav1.2 on microtubule network (scale bar: 5 µm). Bottom panel: Deconvolution of wide-field image of HL-1 cells stained with Cav1.2 (red) and α-tubulin (green). Merged image shows localization of Cav1.2 to the microtubule network. Enlarged pictures (right) indicate that Cav1.2 is distributed along microtubules (## p

    Techniques Used: Expressing, Blocking Assay, Staining

    3) Product Images from "Nuclear CaMKII enhances histone H3 phosphorylation and remodels chromatin during cardiac hypertrophy"

    Article Title: Nuclear CaMKII enhances histone H3 phosphorylation and remodels chromatin during cardiac hypertrophy

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt500

    Increased histone H3 phosphorylation during hypertrophic stimulation. ( A ) Western analysis from primary cardiac cells stimulated with PE and XY using anti-H3 Ser-10, p-CaMKII, α-actinin and GAPDH antibodies. This experiment was repeated three times with three independent cardiomyocyte preparations. ( B ) Quantitation of A. SF: cells maintained in serum-free conditions. ( C ) Western blot analysis of cell extracts prepared from primary cardiomyocytes treated with PE and transfected with control siRNA (siCt) or siRNA against CaMKIIδB (siδB), using α-histone H3 Ser-10 antibody. NT: non-transfected cells ( n = 3). ( D ) Specific silencing of CaMKIIδB by siRNA decreases H3 Ser-10 in ventricular myocytes undergoing hypertrophy. Merge image showing decreased histone H3 phosphorylation in ventricular myocytes stimulated with PE for 24 h transfected with siCaMKIIδB compared with siControl using indirect immunofluorescence. Nuclei are shown in blue with 4′,6-diamidino-2-phenylindole (DAPI) staining, histone H3 Ser-10 is in green. Ventricular cardiomyocytes stained with α-actinin antibody are shown in red. Arrows indicate the nuclei of myocytes positive for H3 Ser-10 in siControl myocytes and arrowheads nuclei which are negative for H3 Ser-10 in ventricular myocytes after CaMKIIδB knock-down. ( E ) Percentage of ventricular myocytes positive for H3 Ser-10. Quantitative analysis of B was done in five different fields. * P
    Figure Legend Snippet: Increased histone H3 phosphorylation during hypertrophic stimulation. ( A ) Western analysis from primary cardiac cells stimulated with PE and XY using anti-H3 Ser-10, p-CaMKII, α-actinin and GAPDH antibodies. This experiment was repeated three times with three independent cardiomyocyte preparations. ( B ) Quantitation of A. SF: cells maintained in serum-free conditions. ( C ) Western blot analysis of cell extracts prepared from primary cardiomyocytes treated with PE and transfected with control siRNA (siCt) or siRNA against CaMKIIδB (siδB), using α-histone H3 Ser-10 antibody. NT: non-transfected cells ( n = 3). ( D ) Specific silencing of CaMKIIδB by siRNA decreases H3 Ser-10 in ventricular myocytes undergoing hypertrophy. Merge image showing decreased histone H3 phosphorylation in ventricular myocytes stimulated with PE for 24 h transfected with siCaMKIIδB compared with siControl using indirect immunofluorescence. Nuclei are shown in blue with 4′,6-diamidino-2-phenylindole (DAPI) staining, histone H3 Ser-10 is in green. Ventricular cardiomyocytes stained with α-actinin antibody are shown in red. Arrows indicate the nuclei of myocytes positive for H3 Ser-10 in siControl myocytes and arrowheads nuclei which are negative for H3 Ser-10 in ventricular myocytes after CaMKIIδB knock-down. ( E ) Percentage of ventricular myocytes positive for H3 Ser-10. Quantitative analysis of B was done in five different fields. * P

    Techniques Used: Western Blot, Quantitation Assay, Transfection, Immunofluorescence, Staining

    CaMKIIδB binds to cardiac chromatin in vitro and in primary cardiomyocytes. ( A ) Coomassie staining of proteins that interact with inactive HA-tagged CaMKIIδB-K43A in cardiac cells, after immunoprecipitation of nuclear extracts using HA agarose affinity gels and analysis by SDS–PAGE. Arrows indicate CaMKIIδB and the histone proteins later identified by mass spectrometry that co-migrate with purified histone octamers. Asterisks show immunoprecipitated proteins at 150 mM salt concentration in cells expressing CaMKIIδB-K43A or GFP. ( B ) Western blot analysis showing interaction of CaMKIIδB-K43A with histone proteins after immunoprecipitation with HA affinity gels ( n = 3). Purified histone octamers were run in parallel as a positive control. ( C ) Interaction of endogenous histone proteins with endogenous CaMKIIδB shown after immunoprecipitation of cardiac nuclear extracts with α-H3 or control IgG antibodies and immunoblotting using a CaMKIIδ-specific antibody. ( D ) Reverse experiment where CaMKIIδ was immunoprecipitated first with anti-CaMKIIδ antibody, and the precipitated proteins were analyzed by SDS–PAGE and immunoblotted with α-H3-specific antibody. Blots in C and D are representative of two independent experiments. ( E ) Western blot analysis showing the fraction of cardio-chromatin that binds to CaMKIIδB attached to HA agarose or that remains in the supernatant ( n = 4). Immunoblotting with anti-H2B and -HA antibodies from 1: input chromatin, 2: chromatin bound to HA-CaMKIIδB, 3: chromatin remaining in the supernatant not bound to HA-CaMKIIδB, 4: chromatin bound to HA beads alone, 5: chromatin remaining in the supernatant not attached to HA beads. ( F ) Representative EMSA with increasing concentration of purified CaMKIIδB and 0W0 or 0W47 mononucleosome templates followed by native PAGE, n = 3. Arrows indicates the position of ISWI or CaMKIIδB bound to mononucleosomes.
    Figure Legend Snippet: CaMKIIδB binds to cardiac chromatin in vitro and in primary cardiomyocytes. ( A ) Coomassie staining of proteins that interact with inactive HA-tagged CaMKIIδB-K43A in cardiac cells, after immunoprecipitation of nuclear extracts using HA agarose affinity gels and analysis by SDS–PAGE. Arrows indicate CaMKIIδB and the histone proteins later identified by mass spectrometry that co-migrate with purified histone octamers. Asterisks show immunoprecipitated proteins at 150 mM salt concentration in cells expressing CaMKIIδB-K43A or GFP. ( B ) Western blot analysis showing interaction of CaMKIIδB-K43A with histone proteins after immunoprecipitation with HA affinity gels ( n = 3). Purified histone octamers were run in parallel as a positive control. ( C ) Interaction of endogenous histone proteins with endogenous CaMKIIδB shown after immunoprecipitation of cardiac nuclear extracts with α-H3 or control IgG antibodies and immunoblotting using a CaMKIIδ-specific antibody. ( D ) Reverse experiment where CaMKIIδ was immunoprecipitated first with anti-CaMKIIδ antibody, and the precipitated proteins were analyzed by SDS–PAGE and immunoblotted with α-H3-specific antibody. Blots in C and D are representative of two independent experiments. ( E ) Western blot analysis showing the fraction of cardio-chromatin that binds to CaMKIIδB attached to HA agarose or that remains in the supernatant ( n = 4). Immunoblotting with anti-H2B and -HA antibodies from 1: input chromatin, 2: chromatin bound to HA-CaMKIIδB, 3: chromatin remaining in the supernatant not bound to HA-CaMKIIδB, 4: chromatin bound to HA beads alone, 5: chromatin remaining in the supernatant not attached to HA beads. ( F ) Representative EMSA with increasing concentration of purified CaMKIIδB and 0W0 or 0W47 mononucleosome templates followed by native PAGE, n = 3. Arrows indicates the position of ISWI or CaMKIIδB bound to mononucleosomes.

    Techniques Used: In Vitro, Staining, Immunoprecipitation, SDS Page, Mass Spectrometry, Purification, Concentration Assay, Expressing, Western Blot, Positive Control, Clear Native PAGE

    H3 Ser-10 phosphorylation in ventricular myocytes is not associated with cellular proliferation. ( A ) Expression level of cell cycle proteins and cycle-dependent kinases during hypertrophic stimulation. Immunoblots from primary cardiac cells stimulated with PE and XY using anti-Cdk4, cyclin E, Cdc2 and GAPDH antibodies. This experiment was repeated twice in two independent cardiomyocyte preparations. SF: serum-free conditions. ( B ) Immunocytochemistry for BrdU and immunofluorescence for Ki67 in primary neonatal rat cardiomyocytes treated with PE for 24 h after transfection with siControl or siCaMKIIδB. CS: cardiomyocytes treated with serum. For Ki67, nuclei are shown in blue with DAPI staining, Ki67 is shown in red. Ventricular cardiomyocytes stained with α-actinin antibody are shown in green.
    Figure Legend Snippet: H3 Ser-10 phosphorylation in ventricular myocytes is not associated with cellular proliferation. ( A ) Expression level of cell cycle proteins and cycle-dependent kinases during hypertrophic stimulation. Immunoblots from primary cardiac cells stimulated with PE and XY using anti-Cdk4, cyclin E, Cdc2 and GAPDH antibodies. This experiment was repeated twice in two independent cardiomyocyte preparations. SF: serum-free conditions. ( B ) Immunocytochemistry for BrdU and immunofluorescence for Ki67 in primary neonatal rat cardiomyocytes treated with PE for 24 h after transfection with siControl or siCaMKIIδB. CS: cardiomyocytes treated with serum. For Ki67, nuclei are shown in blue with DAPI staining, Ki67 is shown in red. Ventricular cardiomyocytes stained with α-actinin antibody are shown in green.

    Techniques Used: Expressing, Western Blot, Immunocytochemistry, Immunofluorescence, Transfection, Staining

    Increased histone H3 Ser-10 and CaMKIIδB recruitment at fetal-cardiac promoters during hypertrophic stimulation. ( A ) Primers used for the amplification of hypertrophic genes after ChIP from primary cardiomyocytes stimulated with PE. Primer sets are specific for the ANF, β-MHC, α-cardiac actin and GAPDH genes. Black boxes represent the position of the primers relative to the start of transcription. ChIP–Q-PCR assay using ( B ) α-histone H3 Ser-10 or ( C ) CaMKIIδ antibodies to precipitate chromatin from primary neonatal rat cardiomyocytes maintained in serum-free conditions or treated with PE for 6, 24 and 24 h. Results are expressed as % input over basal condition (serum-free treatment). Error bars represent means ± SD ( n = 3). ** P
    Figure Legend Snippet: Increased histone H3 Ser-10 and CaMKIIδB recruitment at fetal-cardiac promoters during hypertrophic stimulation. ( A ) Primers used for the amplification of hypertrophic genes after ChIP from primary cardiomyocytes stimulated with PE. Primer sets are specific for the ANF, β-MHC, α-cardiac actin and GAPDH genes. Black boxes represent the position of the primers relative to the start of transcription. ChIP–Q-PCR assay using ( B ) α-histone H3 Ser-10 or ( C ) CaMKIIδ antibodies to precipitate chromatin from primary neonatal rat cardiomyocytes maintained in serum-free conditions or treated with PE for 6, 24 and 24 h. Results are expressed as % input over basal condition (serum-free treatment). Error bars represent means ± SD ( n = 3). ** P

    Techniques Used: Amplification, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    CaMKIIδB specifically phosphorylates histone H3 in histone octamers and in chromatin. ( A ) In vitro kinase assay ( n = 3) with purified constitutively active CaMKIIδB-T287D and reconstituted histone octamers in the presence of [γ 32 P] ATP followed by SDS–PAGE and autoradiography. Asterisks indicate phosphorylation of histone H3 by CaMKIIδB. ( B ) In vitro kinase assays with constitutively active CaMKIIδB-T287D and de-phosphorylated chromatin after treatment with calf intestine alkaline phosphatase (CIAP), purified from neonatal rat cardiomyocytes and HeLa cells in the presence of [γ 32 P] ATP followed by SDS–PAGE ( n = 5). Asterisks indicate phosphorylated histone H3. Lane 1: input de-phosphorylated cardio-chromatin, 2: input de-phosphorylated HeLa chromatin, 3: CaMKIIδB-T287D alone, 4: CaMKIIδB-T287D and de-phosphorylated cardio-chromatin, 5: CaMKIIδB-T287D and de-phosphorylated HeLa chromatin. ( C ) Western blot analysis with phospho-H3 (Ser-10), phospho-H3 (Ser-28) or total H3-specific antibodies after in vitro kinase assay with recombinant histone H3 and active CaMKIIδB-T287D ( n = 3). ( D ) Immunoblots after in vitro phosphorylation assay with endogenous cardio-chromatin or chromatin de-phosphorylated by CIAP treatment in the presence of increasing amount of CaMKIIδB-T287D enzyme, using anti-H3 Ser-10, -H2B and -HA antibodies ( n = 3). Lane 1: cardio-chromatin alone, 2: cardio-chromatin treated with CIAP, 3–5: cardio-chromatin treated with CIAP incubated with different amounts of CaMKIIδB-T287D. ( E ) In vitro kinase assay in the presence of [γ 32 P] ATP after transfection of HeLa cells with CaMKIIδB-T287D and wild-type Flag-histone H3 or mutant histone H3 S10A and immunoprecipitation of isolated chromatin using Flag resins ( n = 3). Lane 1: Flag-H3 wild-type alone, 2: Flag-H3-S10A alone, 3: CaMKIIδB-T287D alone, 4: Flag-H3 wild-type + CaMKIIδB-T287D, 5: Flag-H3-S10A + CaMKIIδB-T287D. ( F ) Immunoblots showing phosphorylation at Ser-10 of wild-type Flag-histone H3 and three different H3 S10A mutants after transfection in HeLa cells and immunoprecipitation using Flag resins ( n = 3). Lane 1: Flag H3 wild-type, 2: Flag-H3 wild-type treated with CIAP, 3–5: Flag-H3-S10A from three separate clones.
    Figure Legend Snippet: CaMKIIδB specifically phosphorylates histone H3 in histone octamers and in chromatin. ( A ) In vitro kinase assay ( n = 3) with purified constitutively active CaMKIIδB-T287D and reconstituted histone octamers in the presence of [γ 32 P] ATP followed by SDS–PAGE and autoradiography. Asterisks indicate phosphorylation of histone H3 by CaMKIIδB. ( B ) In vitro kinase assays with constitutively active CaMKIIδB-T287D and de-phosphorylated chromatin after treatment with calf intestine alkaline phosphatase (CIAP), purified from neonatal rat cardiomyocytes and HeLa cells in the presence of [γ 32 P] ATP followed by SDS–PAGE ( n = 5). Asterisks indicate phosphorylated histone H3. Lane 1: input de-phosphorylated cardio-chromatin, 2: input de-phosphorylated HeLa chromatin, 3: CaMKIIδB-T287D alone, 4: CaMKIIδB-T287D and de-phosphorylated cardio-chromatin, 5: CaMKIIδB-T287D and de-phosphorylated HeLa chromatin. ( C ) Western blot analysis with phospho-H3 (Ser-10), phospho-H3 (Ser-28) or total H3-specific antibodies after in vitro kinase assay with recombinant histone H3 and active CaMKIIδB-T287D ( n = 3). ( D ) Immunoblots after in vitro phosphorylation assay with endogenous cardio-chromatin or chromatin de-phosphorylated by CIAP treatment in the presence of increasing amount of CaMKIIδB-T287D enzyme, using anti-H3 Ser-10, -H2B and -HA antibodies ( n = 3). Lane 1: cardio-chromatin alone, 2: cardio-chromatin treated with CIAP, 3–5: cardio-chromatin treated with CIAP incubated with different amounts of CaMKIIδB-T287D. ( E ) In vitro kinase assay in the presence of [γ 32 P] ATP after transfection of HeLa cells with CaMKIIδB-T287D and wild-type Flag-histone H3 or mutant histone H3 S10A and immunoprecipitation of isolated chromatin using Flag resins ( n = 3). Lane 1: Flag-H3 wild-type alone, 2: Flag-H3-S10A alone, 3: CaMKIIδB-T287D alone, 4: Flag-H3 wild-type + CaMKIIδB-T287D, 5: Flag-H3-S10A + CaMKIIδB-T287D. ( F ) Immunoblots showing phosphorylation at Ser-10 of wild-type Flag-histone H3 and three different H3 S10A mutants after transfection in HeLa cells and immunoprecipitation using Flag resins ( n = 3). Lane 1: Flag H3 wild-type, 2: Flag-H3 wild-type treated with CIAP, 3–5: Flag-H3-S10A from three separate clones.

    Techniques Used: In Vitro, Kinase Assay, Purification, SDS Page, Autoradiography, Western Blot, Recombinant, Phosphorylation Assay, Incubation, Transfection, Mutagenesis, Immunoprecipitation, Isolation, Clone Assay

    4) Product Images from "Nuclear CaMKII enhances histone H3 phosphorylation and remodels chromatin during cardiac hypertrophy"

    Article Title: Nuclear CaMKII enhances histone H3 phosphorylation and remodels chromatin during cardiac hypertrophy

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt500

    Increased histone H3 phosphorylation during hypertrophic stimulation. ( A ) Western analysis from primary cardiac cells stimulated with PE and XY using anti-H3 Ser-10, p-CaMKII, α-actinin and GAPDH antibodies. This experiment was repeated three times with three independent cardiomyocyte preparations. ( B ) Quantitation of A. SF: cells maintained in serum-free conditions. ( C ) Western blot analysis of cell extracts prepared from primary cardiomyocytes treated with PE and transfected with control siRNA (siCt) or siRNA against CaMKIIδB (siδB), using α-histone H3 Ser-10 antibody. NT: non-transfected cells ( n = 3). ( D ) Specific silencing of CaMKIIδB by siRNA decreases H3 Ser-10 in ventricular myocytes undergoing hypertrophy. Merge image showing decreased histone H3 phosphorylation in ventricular myocytes stimulated with PE for 24 h transfected with siCaMKIIδB compared with siControl using indirect immunofluorescence. Nuclei are shown in blue with 4′,6-diamidino-2-phenylindole (DAPI) staining, histone H3 Ser-10 is in green. Ventricular cardiomyocytes stained with α-actinin antibody are shown in red. Arrows indicate the nuclei of myocytes positive for H3 Ser-10 in siControl myocytes and arrowheads nuclei which are negative for H3 Ser-10 in ventricular myocytes after CaMKIIδB knock-down. ( E ) Percentage of ventricular myocytes positive for H3 Ser-10. Quantitative analysis of B was done in five different fields. * P
    Figure Legend Snippet: Increased histone H3 phosphorylation during hypertrophic stimulation. ( A ) Western analysis from primary cardiac cells stimulated with PE and XY using anti-H3 Ser-10, p-CaMKII, α-actinin and GAPDH antibodies. This experiment was repeated three times with three independent cardiomyocyte preparations. ( B ) Quantitation of A. SF: cells maintained in serum-free conditions. ( C ) Western blot analysis of cell extracts prepared from primary cardiomyocytes treated with PE and transfected with control siRNA (siCt) or siRNA against CaMKIIδB (siδB), using α-histone H3 Ser-10 antibody. NT: non-transfected cells ( n = 3). ( D ) Specific silencing of CaMKIIδB by siRNA decreases H3 Ser-10 in ventricular myocytes undergoing hypertrophy. Merge image showing decreased histone H3 phosphorylation in ventricular myocytes stimulated with PE for 24 h transfected with siCaMKIIδB compared with siControl using indirect immunofluorescence. Nuclei are shown in blue with 4′,6-diamidino-2-phenylindole (DAPI) staining, histone H3 Ser-10 is in green. Ventricular cardiomyocytes stained with α-actinin antibody are shown in red. Arrows indicate the nuclei of myocytes positive for H3 Ser-10 in siControl myocytes and arrowheads nuclei which are negative for H3 Ser-10 in ventricular myocytes after CaMKIIδB knock-down. ( E ) Percentage of ventricular myocytes positive for H3 Ser-10. Quantitative analysis of B was done in five different fields. * P

    Techniques Used: Western Blot, Quantitation Assay, Transfection, Immunofluorescence, Staining

    CaMKIIδB binds to cardiac chromatin in vitro and in primary cardiomyocytes. ( A ) Coomassie staining of proteins that interact with inactive HA-tagged CaMKIIδB-K43A in cardiac cells, after immunoprecipitation of nuclear extracts using HA agarose affinity gels and analysis by SDS–PAGE. Arrows indicate CaMKIIδB and the histone proteins later identified by mass spectrometry that co-migrate with purified histone octamers. Asterisks show immunoprecipitated proteins at 150 mM salt concentration in cells expressing CaMKIIδB-K43A or GFP. ( B ) Western blot analysis showing interaction of CaMKIIδB-K43A with histone proteins after immunoprecipitation with HA affinity gels ( n = 3). Purified histone octamers were run in parallel as a positive control. ( C ) Interaction of endogenous histone proteins with endogenous CaMKIIδB shown after immunoprecipitation of cardiac nuclear extracts with α-H3 or control IgG antibodies and immunoblotting using a CaMKIIδ-specific antibody. ( D ) Reverse experiment where CaMKIIδ was immunoprecipitated first with anti-CaMKIIδ antibody, and the precipitated proteins were analyzed by SDS–PAGE and immunoblotted with α-H3-specific antibody. Blots in C and D are representative of two independent experiments. ( E ) Western blot analysis showing the fraction of cardio-chromatin that binds to CaMKIIδB attached to HA agarose or that remains in the supernatant ( n = 4). Immunoblotting with anti-H2B and -HA antibodies from 1: input chromatin, 2: chromatin bound to HA-CaMKIIδB, 3: chromatin remaining in the supernatant not bound to HA-CaMKIIδB, 4: chromatin bound to HA beads alone, 5: chromatin remaining in the supernatant not attached to HA beads. ( F ) Representative EMSA with increasing concentration of purified CaMKIIδB and 0W0 or 0W47 mononucleosome templates followed by native PAGE, n = 3. Arrows indicates the position of ISWI or CaMKIIδB bound to mononucleosomes.
    Figure Legend Snippet: CaMKIIδB binds to cardiac chromatin in vitro and in primary cardiomyocytes. ( A ) Coomassie staining of proteins that interact with inactive HA-tagged CaMKIIδB-K43A in cardiac cells, after immunoprecipitation of nuclear extracts using HA agarose affinity gels and analysis by SDS–PAGE. Arrows indicate CaMKIIδB and the histone proteins later identified by mass spectrometry that co-migrate with purified histone octamers. Asterisks show immunoprecipitated proteins at 150 mM salt concentration in cells expressing CaMKIIδB-K43A or GFP. ( B ) Western blot analysis showing interaction of CaMKIIδB-K43A with histone proteins after immunoprecipitation with HA affinity gels ( n = 3). Purified histone octamers were run in parallel as a positive control. ( C ) Interaction of endogenous histone proteins with endogenous CaMKIIδB shown after immunoprecipitation of cardiac nuclear extracts with α-H3 or control IgG antibodies and immunoblotting using a CaMKIIδ-specific antibody. ( D ) Reverse experiment where CaMKIIδ was immunoprecipitated first with anti-CaMKIIδ antibody, and the precipitated proteins were analyzed by SDS–PAGE and immunoblotted with α-H3-specific antibody. Blots in C and D are representative of two independent experiments. ( E ) Western blot analysis showing the fraction of cardio-chromatin that binds to CaMKIIδB attached to HA agarose or that remains in the supernatant ( n = 4). Immunoblotting with anti-H2B and -HA antibodies from 1: input chromatin, 2: chromatin bound to HA-CaMKIIδB, 3: chromatin remaining in the supernatant not bound to HA-CaMKIIδB, 4: chromatin bound to HA beads alone, 5: chromatin remaining in the supernatant not attached to HA beads. ( F ) Representative EMSA with increasing concentration of purified CaMKIIδB and 0W0 or 0W47 mononucleosome templates followed by native PAGE, n = 3. Arrows indicates the position of ISWI or CaMKIIδB bound to mononucleosomes.

    Techniques Used: In Vitro, Staining, Immunoprecipitation, SDS Page, Mass Spectrometry, Purification, Concentration Assay, Expressing, Western Blot, Positive Control, Clear Native PAGE

    H3 Ser-10 phosphorylation in ventricular myocytes is not associated with cellular proliferation. ( A ) Expression level of cell cycle proteins and cycle-dependent kinases during hypertrophic stimulation. Immunoblots from primary cardiac cells stimulated with PE and XY using anti-Cdk4, cyclin E, Cdc2 and GAPDH antibodies. This experiment was repeated twice in two independent cardiomyocyte preparations. SF: serum-free conditions. ( B ) Immunocytochemistry for BrdU and immunofluorescence for Ki67 in primary neonatal rat cardiomyocytes treated with PE for 24 h after transfection with siControl or siCaMKIIδB. CS: cardiomyocytes treated with serum. For Ki67, nuclei are shown in blue with DAPI staining, Ki67 is shown in red. Ventricular cardiomyocytes stained with α-actinin antibody are shown in green.
    Figure Legend Snippet: H3 Ser-10 phosphorylation in ventricular myocytes is not associated with cellular proliferation. ( A ) Expression level of cell cycle proteins and cycle-dependent kinases during hypertrophic stimulation. Immunoblots from primary cardiac cells stimulated with PE and XY using anti-Cdk4, cyclin E, Cdc2 and GAPDH antibodies. This experiment was repeated twice in two independent cardiomyocyte preparations. SF: serum-free conditions. ( B ) Immunocytochemistry for BrdU and immunofluorescence for Ki67 in primary neonatal rat cardiomyocytes treated with PE for 24 h after transfection with siControl or siCaMKIIδB. CS: cardiomyocytes treated with serum. For Ki67, nuclei are shown in blue with DAPI staining, Ki67 is shown in red. Ventricular cardiomyocytes stained with α-actinin antibody are shown in green.

    Techniques Used: Expressing, Western Blot, Immunocytochemistry, Immunofluorescence, Transfection, Staining

    Increased histone H3 Ser-10 and CaMKIIδB recruitment at fetal-cardiac promoters during hypertrophic stimulation. ( A ) Primers used for the amplification of hypertrophic genes after ChIP from primary cardiomyocytes stimulated with PE. Primer sets are specific for the ANF, β-MHC, α-cardiac actin and GAPDH genes. Black boxes represent the position of the primers relative to the start of transcription. ChIP–Q-PCR assay using ( B ) α-histone H3 Ser-10 or ( C ) CaMKIIδ antibodies to precipitate chromatin from primary neonatal rat cardiomyocytes maintained in serum-free conditions or treated with PE for 6, 24 and 24 h. Results are expressed as % input over basal condition (serum-free treatment). Error bars represent means ± SD ( n = 3). ** P
    Figure Legend Snippet: Increased histone H3 Ser-10 and CaMKIIδB recruitment at fetal-cardiac promoters during hypertrophic stimulation. ( A ) Primers used for the amplification of hypertrophic genes after ChIP from primary cardiomyocytes stimulated with PE. Primer sets are specific for the ANF, β-MHC, α-cardiac actin and GAPDH genes. Black boxes represent the position of the primers relative to the start of transcription. ChIP–Q-PCR assay using ( B ) α-histone H3 Ser-10 or ( C ) CaMKIIδ antibodies to precipitate chromatin from primary neonatal rat cardiomyocytes maintained in serum-free conditions or treated with PE for 6, 24 and 24 h. Results are expressed as % input over basal condition (serum-free treatment). Error bars represent means ± SD ( n = 3). ** P

    Techniques Used: Amplification, Chromatin Immunoprecipitation, Polymerase Chain Reaction

    CaMKIIδB specifically phosphorylates histone H3 in histone octamers and in chromatin. ( A ) In vitro kinase assay ( n = 3) with purified constitutively active CaMKIIδB-T287D and reconstituted histone octamers in the presence of [γ 32 P] ATP followed by SDS–PAGE and autoradiography. Asterisks indicate phosphorylation of histone H3 by CaMKIIδB. ( B ) In vitro kinase assays with constitutively active CaMKIIδB-T287D and de-phosphorylated chromatin after treatment with calf intestine alkaline phosphatase (CIAP), purified from neonatal rat cardiomyocytes and HeLa cells in the presence of [γ 32 P] ATP followed by SDS–PAGE ( n = 5). Asterisks indicate phosphorylated histone H3. Lane 1: input de-phosphorylated cardio-chromatin, 2: input de-phosphorylated HeLa chromatin, 3: CaMKIIδB-T287D alone, 4: CaMKIIδB-T287D and de-phosphorylated cardio-chromatin, 5: CaMKIIδB-T287D and de-phosphorylated HeLa chromatin. ( C ) Western blot analysis with phospho-H3 (Ser-10), phospho-H3 (Ser-28) or total H3-specific antibodies after in vitro kinase assay with recombinant histone H3 and active CaMKIIδB-T287D ( n = 3). ( D ) Immunoblots after in vitro phosphorylation assay with endogenous cardio-chromatin or chromatin de-phosphorylated by CIAP treatment in the presence of increasing amount of CaMKIIδB-T287D enzyme, using anti-H3 Ser-10, -H2B and -HA antibodies ( n = 3). Lane 1: cardio-chromatin alone, 2: cardio-chromatin treated with CIAP, 3–5: cardio-chromatin treated with CIAP incubated with different amounts of CaMKIIδB-T287D. ( E ) In vitro kinase assay in the presence of [γ 32 P] ATP after transfection of HeLa cells with CaMKIIδB-T287D and wild-type Flag-histone H3 or mutant histone H3 S10A and immunoprecipitation of isolated chromatin using Flag resins ( n = 3). Lane 1: Flag-H3 wild-type alone, 2: Flag-H3-S10A alone, 3: CaMKIIδB-T287D alone, 4: Flag-H3 wild-type + CaMKIIδB-T287D, 5: Flag-H3-S10A + CaMKIIδB-T287D. ( F ) Immunoblots showing phosphorylation at Ser-10 of wild-type Flag-histone H3 and three different H3 S10A mutants after transfection in HeLa cells and immunoprecipitation using Flag resins ( n = 3). Lane 1: Flag H3 wild-type, 2: Flag-H3 wild-type treated with CIAP, 3–5: Flag-H3-S10A from three separate clones.
    Figure Legend Snippet: CaMKIIδB specifically phosphorylates histone H3 in histone octamers and in chromatin. ( A ) In vitro kinase assay ( n = 3) with purified constitutively active CaMKIIδB-T287D and reconstituted histone octamers in the presence of [γ 32 P] ATP followed by SDS–PAGE and autoradiography. Asterisks indicate phosphorylation of histone H3 by CaMKIIδB. ( B ) In vitro kinase assays with constitutively active CaMKIIδB-T287D and de-phosphorylated chromatin after treatment with calf intestine alkaline phosphatase (CIAP), purified from neonatal rat cardiomyocytes and HeLa cells in the presence of [γ 32 P] ATP followed by SDS–PAGE ( n = 5). Asterisks indicate phosphorylated histone H3. Lane 1: input de-phosphorylated cardio-chromatin, 2: input de-phosphorylated HeLa chromatin, 3: CaMKIIδB-T287D alone, 4: CaMKIIδB-T287D and de-phosphorylated cardio-chromatin, 5: CaMKIIδB-T287D and de-phosphorylated HeLa chromatin. ( C ) Western blot analysis with phospho-H3 (Ser-10), phospho-H3 (Ser-28) or total H3-specific antibodies after in vitro kinase assay with recombinant histone H3 and active CaMKIIδB-T287D ( n = 3). ( D ) Immunoblots after in vitro phosphorylation assay with endogenous cardio-chromatin or chromatin de-phosphorylated by CIAP treatment in the presence of increasing amount of CaMKIIδB-T287D enzyme, using anti-H3 Ser-10, -H2B and -HA antibodies ( n = 3). Lane 1: cardio-chromatin alone, 2: cardio-chromatin treated with CIAP, 3–5: cardio-chromatin treated with CIAP incubated with different amounts of CaMKIIδB-T287D. ( E ) In vitro kinase assay in the presence of [γ 32 P] ATP after transfection of HeLa cells with CaMKIIδB-T287D and wild-type Flag-histone H3 or mutant histone H3 S10A and immunoprecipitation of isolated chromatin using Flag resins ( n = 3). Lane 1: Flag-H3 wild-type alone, 2: Flag-H3-S10A alone, 3: CaMKIIδB-T287D alone, 4: Flag-H3 wild-type + CaMKIIδB-T287D, 5: Flag-H3-S10A + CaMKIIδB-T287D. ( F ) Immunoblots showing phosphorylation at Ser-10 of wild-type Flag-histone H3 and three different H3 S10A mutants after transfection in HeLa cells and immunoprecipitation using Flag resins ( n = 3). Lane 1: Flag H3 wild-type, 2: Flag-H3 wild-type treated with CIAP, 3–5: Flag-H3-S10A from three separate clones.

    Techniques Used: In Vitro, Kinase Assay, Purification, SDS Page, Autoradiography, Western Blot, Recombinant, Phosphorylation Assay, Incubation, Transfection, Mutagenesis, Immunoprecipitation, Isolation, Clone Assay

    5) Product Images from "Biologically active constituents of the secretome of human W8B2+ cardiac stem cells"

    Article Title: Biologically active constituents of the secretome of human W8B2+ cardiac stem cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-19855-4

    Molecular and biological regenerative properties of extracellular vesicle fractions of W8B2 + CSC-conditioned medium. ( A ) Particle size distribution shows extracellular vesicles with an average modal size of 201 ± 12 nm. ( B ) Electron micrograph images of extracellular vesicles shows near-spherical shape of double membraned vesicles. ( C ) DELFIA protein analysis shows the expression of CD81 in the extracellular vesicles isolated from 2 different biological samples. The effect of extracellular vesicles on survival of ( D ) neonatal rat cardiomyocytes and ( E ) human cardiac microvascular endothelial cells in culture after simulated ischaemia. The effect of extracellular vesicles on ( F ) proliferation of neonatal rat cardiomyocytes proliferation and ( G ) pro-angiogenic tube formation of human cardiac microvascular endothelial cells, assessed by the number of loops formed. Data are shown as mean ± SEM from 4–6 independent experiments. *p
    Figure Legend Snippet: Molecular and biological regenerative properties of extracellular vesicle fractions of W8B2 + CSC-conditioned medium. ( A ) Particle size distribution shows extracellular vesicles with an average modal size of 201 ± 12 nm. ( B ) Electron micrograph images of extracellular vesicles shows near-spherical shape of double membraned vesicles. ( C ) DELFIA protein analysis shows the expression of CD81 in the extracellular vesicles isolated from 2 different biological samples. The effect of extracellular vesicles on survival of ( D ) neonatal rat cardiomyocytes and ( E ) human cardiac microvascular endothelial cells in culture after simulated ischaemia. The effect of extracellular vesicles on ( F ) proliferation of neonatal rat cardiomyocytes proliferation and ( G ) pro-angiogenic tube formation of human cardiac microvascular endothelial cells, assessed by the number of loops formed. Data are shown as mean ± SEM from 4–6 independent experiments. *p

    Techniques Used: Expressing, Isolation

    Biological activity profile of W8B2 + CSC-conditioned medium fractionated by ion exchange chromatography. The effect of conditioned medium on survival of ( A ) neonatal rat cardiomyocytes and ( B ) human cardiac microvascular endothelial cells. ( C ) Proliferation of neonatal rat cardiomyocytes represented by percentage of Ki67 + cells. ( D ) Pro-angiogenic tube formation of human cardiac microvascular endothelial cells, assessed by the number of loops formed in vitro . Serum-free medium and serum-free medium supplemented with 5% FCS (FCS) were served as Control and as a positive control (FCS), respectively. Data are shown as mean ± SEM from 4–6 independent experiments. *p
    Figure Legend Snippet: Biological activity profile of W8B2 + CSC-conditioned medium fractionated by ion exchange chromatography. The effect of conditioned medium on survival of ( A ) neonatal rat cardiomyocytes and ( B ) human cardiac microvascular endothelial cells. ( C ) Proliferation of neonatal rat cardiomyocytes represented by percentage of Ki67 + cells. ( D ) Pro-angiogenic tube formation of human cardiac microvascular endothelial cells, assessed by the number of loops formed in vitro . Serum-free medium and serum-free medium supplemented with 5% FCS (FCS) were served as Control and as a positive control (FCS), respectively. Data are shown as mean ± SEM from 4–6 independent experiments. *p

    Techniques Used: Activity Assay, Ion Exchange Chromatography, In Vitro, Positive Control

    6) Product Images from "ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression"

    Article Title: ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-31784-w

    ADAP1 does not interfere with Mek1ca-induced fetal gene program activation. ( A – I ) Analysis by RT-qPCR of different mRNA expressed in rat neonatal ventricular cardiomyocytes (RNVC) that are representative of the fetal gene program. The RNVC were infected with an β-Gal- (negative control), ADAP1-, or Mek1ca-overexpressing adenovirus, individually or in combination as indicated, and were cultured for 72 h post-infection. The histograms represent mRNA expression levels relative to the β-Gal control and normalized to the Rpl30 reporter gene ( n = 3 independent experiments). * P
    Figure Legend Snippet: ADAP1 does not interfere with Mek1ca-induced fetal gene program activation. ( A – I ) Analysis by RT-qPCR of different mRNA expressed in rat neonatal ventricular cardiomyocytes (RNVC) that are representative of the fetal gene program. The RNVC were infected with an β-Gal- (negative control), ADAP1-, or Mek1ca-overexpressing adenovirus, individually or in combination as indicated, and were cultured for 72 h post-infection. The histograms represent mRNA expression levels relative to the β-Gal control and normalized to the Rpl30 reporter gene ( n = 3 independent experiments). * P

    Techniques Used: Activation Assay, Quantitative RT-PCR, Infection, Negative Control, Cell Culture, Expressing

    ADAP1 restrains the serum-induced increase in cell size of cultured cardiomyocytes. ( A ) Western blot detection of adenovirus-mediated 3xFLAG-hADAP1 overexpression (MOI of 50) in rat neonatal ventricular cardiomyocytes (RNVC) cultured for 72 h post-infection. ( B ) RNVC were infected with either β-Gal- (negative control) or ADAP1-overexpressing adenovirus and were cultured for 72 h in the absence (0%) or presence (10%) of serum. Representative images of α-Actinin-immunostained RNVC (left) and corresponding segmented images were acquired using the Operetta High-Content Imaging System (Perkin Elmer). The scale bar represents 50 µm. ( C ) The histogram represents the cell surface areas of RNVC overexpressing either β-Gal or ADAP1 and cultured for 72 h with increasing concentrations of serum ( n = 3 independent experiments). * P
    Figure Legend Snippet: ADAP1 restrains the serum-induced increase in cell size of cultured cardiomyocytes. ( A ) Western blot detection of adenovirus-mediated 3xFLAG-hADAP1 overexpression (MOI of 50) in rat neonatal ventricular cardiomyocytes (RNVC) cultured for 72 h post-infection. ( B ) RNVC were infected with either β-Gal- (negative control) or ADAP1-overexpressing adenovirus and were cultured for 72 h in the absence (0%) or presence (10%) of serum. Representative images of α-Actinin-immunostained RNVC (left) and corresponding segmented images were acquired using the Operetta High-Content Imaging System (Perkin Elmer). The scale bar represents 50 µm. ( C ) The histogram represents the cell surface areas of RNVC overexpressing either β-Gal or ADAP1 and cultured for 72 h with increasing concentrations of serum ( n = 3 independent experiments). * P

    Techniques Used: Cell Culture, Western Blot, Over Expression, Infection, Negative Control, Imaging

    ADAP1 blocks phenylephrine- and Mek1ca-induced hypertrophy. Cell surface area measurements of rat neonatal ventricular cardiomyocytes (RNVC) overexpressing ADAP1 in the absence or presence of 50 µM phenylephrine ( A ) or Mek1ca-overexpressing adenovirus ( B ) compared with an adβ-Gal-infected control. Quantification of at least 3 independent experiments expressed as means ± SD. * P
    Figure Legend Snippet: ADAP1 blocks phenylephrine- and Mek1ca-induced hypertrophy. Cell surface area measurements of rat neonatal ventricular cardiomyocytes (RNVC) overexpressing ADAP1 in the absence or presence of 50 µM phenylephrine ( A ) or Mek1ca-overexpressing adenovirus ( B ) compared with an adβ-Gal-infected control. Quantification of at least 3 independent experiments expressed as means ± SD. * P

    Techniques Used: Infection

    ADAP1 relocalizes cytoskeletal α-Actinin. ( A ) Representative confocal images (Olympus FluoView FV1000 microscope) of α-Actinin-immunostained rat neonatal ventricular cardiomyocytes (RNVC) infected with a β-Gal- (negative control), ADAP1-, or Mek1ca-overexpressing adenovirus, individually or in combination as indicated, and cultured for 72 h post-infection. Arrows point to α-Actinin dense puncta. The scale bar represents 12 µm. ( B ) Number of α-Actinin puncta per cell measured with the Operetta High-Content Imaging System (Perkin Elmer) using the same experimental conditions as in A ( n = 4 independent experiments). **** P
    Figure Legend Snippet: ADAP1 relocalizes cytoskeletal α-Actinin. ( A ) Representative confocal images (Olympus FluoView FV1000 microscope) of α-Actinin-immunostained rat neonatal ventricular cardiomyocytes (RNVC) infected with a β-Gal- (negative control), ADAP1-, or Mek1ca-overexpressing adenovirus, individually or in combination as indicated, and cultured for 72 h post-infection. Arrows point to α-Actinin dense puncta. The scale bar represents 12 µm. ( B ) Number of α-Actinin puncta per cell measured with the Operetta High-Content Imaging System (Perkin Elmer) using the same experimental conditions as in A ( n = 4 independent experiments). **** P

    Techniques Used: Microscopy, Infection, Negative Control, Cell Culture, Imaging

    Adap1 expression in cardiac cells. ( A ) Relative levels of Adap1 mRNA expression in the rat brain (adult) and heart (adult and 2-day-old neonate) were measured by RT-qPCR and were normalized to the Rpl30 reporter gene ( n = 4 independent tissues). ( B ) Representative Western blots of Adap1 and Gapdh (loading control) detected from whole brain and heart extracts. ( C ) The histogram represents the relative expression level of Adap1 normalized to Gapdh in the respective tissues ( n = 4 independent tissues). ( D ) Representative Western blot of Adap1, α-Actinin (cardiomyocyte specific marker), and Gapdh (loading control) detected in the protein lysates of enriched rat neonatal ventricular cardiomyocytes (RNVC) and non-cardiomyocytes (Non-CM). ( E ) The histogram represents the relative expression level of Adap1 normalized to Gapdh in the respective cell lysates ( n .
    Figure Legend Snippet: Adap1 expression in cardiac cells. ( A ) Relative levels of Adap1 mRNA expression in the rat brain (adult) and heart (adult and 2-day-old neonate) were measured by RT-qPCR and were normalized to the Rpl30 reporter gene ( n = 4 independent tissues). ( B ) Representative Western blots of Adap1 and Gapdh (loading control) detected from whole brain and heart extracts. ( C ) The histogram represents the relative expression level of Adap1 normalized to Gapdh in the respective tissues ( n = 4 independent tissues). ( D ) Representative Western blot of Adap1, α-Actinin (cardiomyocyte specific marker), and Gapdh (loading control) detected in the protein lysates of enriched rat neonatal ventricular cardiomyocytes (RNVC) and non-cardiomyocytes (Non-CM). ( E ) The histogram represents the relative expression level of Adap1 normalized to Gapdh in the respective cell lysates ( n .

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Marker

    7) Product Images from "Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency"

    Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.06.002

    The LINC00116 -Derived Microprotein Mitoregulin Localizes to Inner Mitochondrial Membranes and Binds Cardiolipin (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.
    Figure Legend Snippet: The LINC00116 -Derived Microprotein Mitoregulin Localizes to Inner Mitochondrial Membranes and Binds Cardiolipin (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.

    Techniques Used: Derivative Assay, Isolation, Knock-Out, Western Blot, Pull Down Assay, Binding Assay, Positive Control, Stripping Membranes

    8) Product Images from "Depletion of PHD3 Protects Heart from Ischemia/Reperfusion Injury by Inhibiting Cardiomyocyte Apoptosis"

    Article Title: Depletion of PHD3 Protects Heart from Ischemia/Reperfusion Injury by Inhibiting Cardiomyocyte Apoptosis

    Journal: Journal of molecular and cellular cardiology

    doi: 10.1016/j.yjmcc.2015.01.007

    PHD3 plays a crucial role in DNA damage response and apoptosis induced by H 2 O 2 or hypoxia-reoxygenation in cardiomyocytes (A) HL-1 cells were pre-treated with DMOG for 4h or KU55933 for 30 minutes and then treated with 100 μM or 200 μM H 2 O 2 for 1 hour as indicated. Western blots were performed with the indicated antibodies. (B) HL-1 cells were cultured in a hypoxia chamber for 6 hours and then switched to normoxic conditions for the indicated time with or without pretreatment with DMOG. Western blots were performed with the indicated antibodies. (C) Neonatal mouse ventricular myocytes (NMVMs) from PHD3 f / f ; Cre +/− or PHD3 f / f ; Cre −/− mice were treated with 4-hydroxyl-tamoxifen for 3 days. Cells were then treated with NCS, Doxorubicin or H 2 O 2 for 1h and western blots were performed with indicated antibodies. (D), (E) NMVMs from PHD3 f / f mice were infected with adenovirus expressing cre recombinase or lacZ for 2 days. Infected cells were then cultured in ischemic medium at hypoxic condition for 1 hour. Re-oxygenation was obtained by culturing cells in normal medium at normoxic condition for 16 hours. Cells were immunostained with MF20 and apoptosis was analyzed with TUNEL staining. Quantitative analysis was shown in (E) . n=3, *p
    Figure Legend Snippet: PHD3 plays a crucial role in DNA damage response and apoptosis induced by H 2 O 2 or hypoxia-reoxygenation in cardiomyocytes (A) HL-1 cells were pre-treated with DMOG for 4h or KU55933 for 30 minutes and then treated with 100 μM or 200 μM H 2 O 2 for 1 hour as indicated. Western blots were performed with the indicated antibodies. (B) HL-1 cells were cultured in a hypoxia chamber for 6 hours and then switched to normoxic conditions for the indicated time with or without pretreatment with DMOG. Western blots were performed with the indicated antibodies. (C) Neonatal mouse ventricular myocytes (NMVMs) from PHD3 f / f ; Cre +/− or PHD3 f / f ; Cre −/− mice were treated with 4-hydroxyl-tamoxifen for 3 days. Cells were then treated with NCS, Doxorubicin or H 2 O 2 for 1h and western blots were performed with indicated antibodies. (D), (E) NMVMs from PHD3 f / f mice were infected with adenovirus expressing cre recombinase or lacZ for 2 days. Infected cells were then cultured in ischemic medium at hypoxic condition for 1 hour. Re-oxygenation was obtained by culturing cells in normal medium at normoxic condition for 16 hours. Cells were immunostained with MF20 and apoptosis was analyzed with TUNEL staining. Quantitative analysis was shown in (E) . n=3, *p

    Techniques Used: Western Blot, Cell Culture, Mouse Assay, Infection, Expressing, TUNEL Assay, Staining

    DMOG inhibits DNA damage response and apoptosis induced by doxorubicin in primary cardiomyocytes (A) Neonatal rat ventricular myocytes were pre-treated with DMOG for 4h and then treated with doxorubicin (1μM) as indicated. Western blots were performed with the indicated antibodies. (B), (C) Neonatal rat ventricular myocytes were treated with doxorubicin for 16h with or without pretreatment of DMOG. Cardiomyocyte apoptosis was then analyzed with TUNEL staining. Neonatal rat ventricular myocytes were also immunostained with MF20 antibody, which specifically recognizes myosin of striated muscle cells. Quantitative analysis is from 3 independent experiments. *p
    Figure Legend Snippet: DMOG inhibits DNA damage response and apoptosis induced by doxorubicin in primary cardiomyocytes (A) Neonatal rat ventricular myocytes were pre-treated with DMOG for 4h and then treated with doxorubicin (1μM) as indicated. Western blots were performed with the indicated antibodies. (B), (C) Neonatal rat ventricular myocytes were treated with doxorubicin for 16h with or without pretreatment of DMOG. Cardiomyocyte apoptosis was then analyzed with TUNEL staining. Neonatal rat ventricular myocytes were also immunostained with MF20 antibody, which specifically recognizes myosin of striated muscle cells. Quantitative analysis is from 3 independent experiments. *p

    Techniques Used: Western Blot, TUNEL Assay, Staining

    Depletion of PHD3 inhibits cardiomyocyte apoptosis induced by I/R injury After 5 doses of tamoxifen infusion, left anterior descending (LAD) coronary arteries of mice with indicated genotypes were tied for 40 minutes and then released for reperfusion. Twenty-four hours after reperfusion, hearts were fixed with 10% formaldehyde and embedded in paraffin. Cross sections of hearts were then analyzed with TUNEL staining. Nuclei were stained with DAPI. Representative high magnification images of the AAR are shown in (A) and low magnification images of whole sections are shown in (B) . Quantitative analysis of the apoptotic cells within the AAR is shown in (C). The numbers of mice analyzed are indicated in the bars respectively. *, p
    Figure Legend Snippet: Depletion of PHD3 inhibits cardiomyocyte apoptosis induced by I/R injury After 5 doses of tamoxifen infusion, left anterior descending (LAD) coronary arteries of mice with indicated genotypes were tied for 40 minutes and then released for reperfusion. Twenty-four hours after reperfusion, hearts were fixed with 10% formaldehyde and embedded in paraffin. Cross sections of hearts were then analyzed with TUNEL staining. Nuclei were stained with DAPI. Representative high magnification images of the AAR are shown in (A) and low magnification images of whole sections are shown in (B) . Quantitative analysis of the apoptotic cells within the AAR is shown in (C). The numbers of mice analyzed are indicated in the bars respectively. *, p

    Techniques Used: Mouse Assay, TUNEL Assay, Staining

    Depletion of PHD3 has no effect on HIF-1α protein level, the expression of HIF target genes or capillary density in the heart After 5 doses of tamoxifen, ventricles were excised and flash frozen. (A) Proteins extracted from ventricles of the indicated genotypes (n=3) were western-blotted with anti-HIF-1α and anti-PHD3 antibodies. Lysate from cardiomyocytes treated with DMOG (1mM) was used as the positive control for HIF-1α. (B) mRNAs were extracted from ventricles of the indicated genotypes. Relative mRNA level of HIF target genes and PHD3 were analyzed by quantitative real-time PCR. n = 3. (C) Heart sections of the indicated genotypes were immunostained with anti-myosin antibody and TRITC-E-lectin. Capillary densities are expressed as the number of lectin-positive objects per field of view. N.S., not significant, n = 3.
    Figure Legend Snippet: Depletion of PHD3 has no effect on HIF-1α protein level, the expression of HIF target genes or capillary density in the heart After 5 doses of tamoxifen, ventricles were excised and flash frozen. (A) Proteins extracted from ventricles of the indicated genotypes (n=3) were western-blotted with anti-HIF-1α and anti-PHD3 antibodies. Lysate from cardiomyocytes treated with DMOG (1mM) was used as the positive control for HIF-1α. (B) mRNAs were extracted from ventricles of the indicated genotypes. Relative mRNA level of HIF target genes and PHD3 were analyzed by quantitative real-time PCR. n = 3. (C) Heart sections of the indicated genotypes were immunostained with anti-myosin antibody and TRITC-E-lectin. Capillary densities are expressed as the number of lectin-positive objects per field of view. N.S., not significant, n = 3.

    Techniques Used: Expressing, Western Blot, Positive Control, Real-time Polymerase Chain Reaction

    Depletion of PHD3 further stabilizes HIF-1α and overexpression of normoxia-stable HIF-1α protects cardiomyocytes from hypoxia-induced apoptosis (A) Neonatal ventricular myocytes from PHD3 f / f ; Cre +/− or PHD3 f / f ; Cre −/− mice were treated with 4-hydroxyl-tamoxifen for 3 days to delete PHD3. Cells were then cultured at 0.5% or 21% O 2 condition for 8 hours. Cells were then harvested for western blots with the indicated antibodies. (B), (C) HL-1 cardiomyocytes were infected with adenovirus expressing normoxia-stable HIF-1α-GFP (HIF-1α PP/AG -GFP) or lacZ for 24 hours. Infected cells were then cultured with fresh serum-free medium at 0.5% or 21% O 2 conditions for additional 48 hours to induce apoptosis. Cardiomyocytes were then fixed and stained with DAPI. Apoptosis was then analyzed with TUNEL staining. *, p
    Figure Legend Snippet: Depletion of PHD3 further stabilizes HIF-1α and overexpression of normoxia-stable HIF-1α protects cardiomyocytes from hypoxia-induced apoptosis (A) Neonatal ventricular myocytes from PHD3 f / f ; Cre +/− or PHD3 f / f ; Cre −/− mice were treated with 4-hydroxyl-tamoxifen for 3 days to delete PHD3. Cells were then cultured at 0.5% or 21% O 2 condition for 8 hours. Cells were then harvested for western blots with the indicated antibodies. (B), (C) HL-1 cardiomyocytes were infected with adenovirus expressing normoxia-stable HIF-1α-GFP (HIF-1α PP/AG -GFP) or lacZ for 24 hours. Infected cells were then cultured with fresh serum-free medium at 0.5% or 21% O 2 conditions for additional 48 hours to induce apoptosis. Cardiomyocytes were then fixed and stained with DAPI. Apoptosis was then analyzed with TUNEL staining. *, p

    Techniques Used: Over Expression, Mouse Assay, Cell Culture, Western Blot, Infection, Expressing, Staining, TUNEL Assay

    DMOG inhibits HL-1 cardiomyocyte apoptosis induced by doxorubicin (A) HL-1 cells were transfected with two sets of si-RNA for Chk1 or scramble si-RNA as the control (Si-C) for two days. Cells were then treated with doxorubicin (1μM) as indicated. Western blots were then performed with the indicated antibodies. (B) After two days transfection with si-RNAs, HL-1 cells were treated with doxorubicin (1μM) for 8 hours and then harvested for caspase3/7 activity assay. Knocking down the expression of Chk-1 significantly inhibits caspase3/7 activity. N = 3, *p
    Figure Legend Snippet: DMOG inhibits HL-1 cardiomyocyte apoptosis induced by doxorubicin (A) HL-1 cells were transfected with two sets of si-RNA for Chk1 or scramble si-RNA as the control (Si-C) for two days. Cells were then treated with doxorubicin (1μM) as indicated. Western blots were then performed with the indicated antibodies. (B) After two days transfection with si-RNAs, HL-1 cells were treated with doxorubicin (1μM) for 8 hours and then harvested for caspase3/7 activity assay. Knocking down the expression of Chk-1 significantly inhibits caspase3/7 activity. N = 3, *p

    Techniques Used: Transfection, Western Blot, Activity Assay, Expressing

    9) Product Images from "DNA single-strand break-induced DNA damage response causes heart failure"

    Article Title: DNA single-strand break-induced DNA damage response causes heart failure

    Journal: Nature Communications

    doi: 10.1038/ncomms15104

    Generation of an in vitro model of cardiomyocytes with SSB accumulation. ( a ) Neonatal rat cardiomyocytes (NRCMs) were treated with MMS at the indicated concentration for 10 min and the DNA damage was analysed by comet assay (Alkaline comet: n =42, 37, 45, 33, 34; Neutral comet: n =40, 35, 35, 37, 29 at each concentration, respectively). Statistical significance was determined by Steel–Dwass test. ## P
    Figure Legend Snippet: Generation of an in vitro model of cardiomyocytes with SSB accumulation. ( a ) Neonatal rat cardiomyocytes (NRCMs) were treated with MMS at the indicated concentration for 10 min and the DNA damage was analysed by comet assay (Alkaline comet: n =42, 37, 45, 33, 34; Neutral comet: n =40, 35, 35, 37, 29 at each concentration, respectively). Statistical significance was determined by Steel–Dwass test. ## P

    Techniques Used: In Vitro, Concentration Assay, Single Cell Gel Electrophoresis

    Possible roles of SSB accumulation in pathogenesis of heart failure. Accumulation of DNA SSB in cardiomyocytes induces persistent activation of DDR and subsequent activation of NF-κB pathway, resulting in increased expressions of inflammatory cytokines. These mechanisms may contribute, at least in part, to increased cardiac inflammation and the progression of pressure overload-induced heart failure.
    Figure Legend Snippet: Possible roles of SSB accumulation in pathogenesis of heart failure. Accumulation of DNA SSB in cardiomyocytes induces persistent activation of DDR and subsequent activation of NF-κB pathway, resulting in increased expressions of inflammatory cytokines. These mechanisms may contribute, at least in part, to increased cardiac inflammation and the progression of pressure overload-induced heart failure.

    Techniques Used: Activation Assay

    Xrcc1 deficiency exacerbates cardiac inflammation after pressure overload. ( a , b ) Activation of DDR in Sham- or TAC-operated Xrcc1 f/f and Xrcc1 αMHC-Cre mice was assessed by immunostaining for phosphorylated H2AX ( a , γH2AX, green). Immunostaining for alpha-actinin (red) was used to label cardiomyocytes. Arrowheads indicate γH2AX-positive cardiomyocytes and arrows indicate γH2AX-positive non-cardiomyocytes. Scale bar, 50 μm. The number of γH2AX-positive cardiomyocytes was counted ( b , n =4 each). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Tukey–Kramer HSD test. ** P
    Figure Legend Snippet: Xrcc1 deficiency exacerbates cardiac inflammation after pressure overload. ( a , b ) Activation of DDR in Sham- or TAC-operated Xrcc1 f/f and Xrcc1 αMHC-Cre mice was assessed by immunostaining for phosphorylated H2AX ( a , γH2AX, green). Immunostaining for alpha-actinin (red) was used to label cardiomyocytes. Arrowheads indicate γH2AX-positive cardiomyocytes and arrows indicate γH2AX-positive non-cardiomyocytes. Scale bar, 50 μm. The number of γH2AX-positive cardiomyocytes was counted ( b , n =4 each). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Tukey–Kramer HSD test. ** P

    Techniques Used: Activation Assay, Mouse Assay, Immunostaining

    SSB activates DDR and induce inflammation through NF-κB. ( a ) Neonatal rat cardiomyocytes (NRCMs) were treated with MMS (0.05 mg ml −1 for 10 min) or vehicle control (Mock) and activation of DDR was assessed by western blotting against phospho- or total ATM, H2AX and p53 at the indicated time point. Western blotting against GAPDH was used as a loading control. ( b ) NRCMs were treated with MMS (0.05 mg ml −1 for 10 min) or vehicle alone (Mock) for 3 consecutive days and activation of DDR was assessed as described in a . ( c ) NRCMs were transfected with siRNA against Xrcc1 (siXrcc1) or scrambled negative control oligonucleotide (Scramble). Four days later, activation of DDR was assessed as described in a . ( d–f ) NRCMs were transfected with siRNA against Xrcc1 and/or Atm. Expression levels of inflammatory cytokines were assessed by real-time PCR ( d , n =6 each, technical duplicates). Nuclear translocation of NF-κB was assessed by immunofluorescence ( e , green). The nuclei of the cells were counterstained with TO-PRO-3 iodide 642/661 (blue). Scale bar, 20 μm. Cells with positive nuclear NF-κB staining were counted ( f , n =7, 8, 8, 5, respectively). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer HSD test for ( d , f ) ** P
    Figure Legend Snippet: SSB activates DDR and induce inflammation through NF-κB. ( a ) Neonatal rat cardiomyocytes (NRCMs) were treated with MMS (0.05 mg ml −1 for 10 min) or vehicle control (Mock) and activation of DDR was assessed by western blotting against phospho- or total ATM, H2AX and p53 at the indicated time point. Western blotting against GAPDH was used as a loading control. ( b ) NRCMs were treated with MMS (0.05 mg ml −1 for 10 min) or vehicle alone (Mock) for 3 consecutive days and activation of DDR was assessed as described in a . ( c ) NRCMs were transfected with siRNA against Xrcc1 (siXrcc1) or scrambled negative control oligonucleotide (Scramble). Four days later, activation of DDR was assessed as described in a . ( d–f ) NRCMs were transfected with siRNA against Xrcc1 and/or Atm. Expression levels of inflammatory cytokines were assessed by real-time PCR ( d , n =6 each, technical duplicates). Nuclear translocation of NF-κB was assessed by immunofluorescence ( e , green). The nuclei of the cells were counterstained with TO-PRO-3 iodide 642/661 (blue). Scale bar, 20 μm. Cells with positive nuclear NF-κB staining were counted ( f , n =7, 8, 8, 5, respectively). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer HSD test for ( d , f ) ** P

    Techniques Used: Activation Assay, Western Blot, Transfection, Negative Control, Expressing, Real-time Polymerase Chain Reaction, Translocation Assay, Immunofluorescence, Staining

    Accumulation of DNA SSB in the failing heart. ( a , b ) Cardiomyocytes were isolated from the TAC-operated heart at the indicated time points. The type of DNA damage in cardiomyocytes was assessed by comet assay. Representative images ( a ) and quantitative analyses are shown ( b , Alkaline comet: n =28, 45, 48; Neutral comet: n =38, 56, 44 at each time point, respectively, biological replicates=3). ( c , d ) Fragmented DNA and DSB were labelled with ISOL staining ( c , green). Wheat germ agglutinin (WGA, red) was used to visualize cardiomyocytes. DNase-treated section (DNase I, 10 Kunitz units ml −1 ) was used as a positive control. Arrowheads indicate ISOL-positive cardiomyocytes and arrows indicate ISOL-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. The number of ISOL-positive cardiomyocytes was counted ( d , n =4 each). ( e , f ) Heart tissue sections were immunostained for NBS1 ( e , NBS1, green). Immunostaining for alpha-actinin (red) was used to label cardiomyocytes. Scale bar, 50 μm. The number of NBS1-positive cardiomyocytes was counted ( f , n =4 each). ( g , h ) Heart tissue sections were immunostained for poly-ADP ribose ( g , PAR, green) and the number of PAR-positive cardiomyocytes was counted ( h , n =4, 4, 5 at each time point, respectively). Arrowheads indicate PAR-positive cardiomyocytes and arrows indicate PAR-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. ( i ) Expression levels of SSB repair enzymes were analysed by real-time PCR ( n =4, 6, 8 at each time point, respectively, technical duplicates). ( j , k ) Heart tissue sections were stained with dihydroethidium ( i , DHE, 10 μΜ) and mean fluorescence intensity relative to Sham-operated mice was measured ( k , n =4, 5, 5 at each time point, respectively). Scale bar, 50 μm. ( l ) The level of H 2 O 2 in the TAC-operated heart was measured using Amplex Red assay ( n =9, 5, 6 at each time point, respectively). Statistical significance was determined by Steel-Dwass test for ( b ) and by one-way analysis of variance followed by the Tukey–Kramer HSD test for ( d , f , h , i , j ) * P
    Figure Legend Snippet: Accumulation of DNA SSB in the failing heart. ( a , b ) Cardiomyocytes were isolated from the TAC-operated heart at the indicated time points. The type of DNA damage in cardiomyocytes was assessed by comet assay. Representative images ( a ) and quantitative analyses are shown ( b , Alkaline comet: n =28, 45, 48; Neutral comet: n =38, 56, 44 at each time point, respectively, biological replicates=3). ( c , d ) Fragmented DNA and DSB were labelled with ISOL staining ( c , green). Wheat germ agglutinin (WGA, red) was used to visualize cardiomyocytes. DNase-treated section (DNase I, 10 Kunitz units ml −1 ) was used as a positive control. Arrowheads indicate ISOL-positive cardiomyocytes and arrows indicate ISOL-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. The number of ISOL-positive cardiomyocytes was counted ( d , n =4 each). ( e , f ) Heart tissue sections were immunostained for NBS1 ( e , NBS1, green). Immunostaining for alpha-actinin (red) was used to label cardiomyocytes. Scale bar, 50 μm. The number of NBS1-positive cardiomyocytes was counted ( f , n =4 each). ( g , h ) Heart tissue sections were immunostained for poly-ADP ribose ( g , PAR, green) and the number of PAR-positive cardiomyocytes was counted ( h , n =4, 4, 5 at each time point, respectively). Arrowheads indicate PAR-positive cardiomyocytes and arrows indicate PAR-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. ( i ) Expression levels of SSB repair enzymes were analysed by real-time PCR ( n =4, 6, 8 at each time point, respectively, technical duplicates). ( j , k ) Heart tissue sections were stained with dihydroethidium ( i , DHE, 10 μΜ) and mean fluorescence intensity relative to Sham-operated mice was measured ( k , n =4, 5, 5 at each time point, respectively). Scale bar, 50 μm. ( l ) The level of H 2 O 2 in the TAC-operated heart was measured using Amplex Red assay ( n =9, 5, 6 at each time point, respectively). Statistical significance was determined by Steel-Dwass test for ( b ) and by one-way analysis of variance followed by the Tukey–Kramer HSD test for ( d , f , h , i , j ) * P

    Techniques Used: Isolation, Single Cell Gel Electrophoresis, Staining, Whole Genome Amplification, Positive Control, Immunostaining, Expressing, Real-time Polymerase Chain Reaction, Fluorescence, Mouse Assay, Amplex Red Assay

    ATM gene deletion rescues the cardiac phenotypes of Xrcc1 deficient mice. ( a , b ) Macroscopic and echocardiographic images ( a ) and cardiac function ( b ) of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice ( Xrcc1 f/f mice: n =83, 21, 46, 11, 27; Xrcc1 αMHC-Cre mice: n =88, 28, 60, 13, 16; Xrcc1 αMHC-Cre ; Atm +/− mice: n =28, 22, 22, 7, 7 at each time point, respectively). Scale bar, 2 mm. ( c ) Heart, lung, and body weight of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice were weighed 8 weeks after the surgery ( n =8, 5, 6 for each genotype, respectively). ( d ) Survival curves of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice ( n =49, 62, 23, respectively). ( e – k ) TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice were analysed 4 weeks after the surgery. The type of DNA damage in cardiomyocytes was assessed by comet assay ( e , Alkaline comet: n =50, 76, 77; Neutral comet: n =53, 56, 42, respectively). Activation of DDR was assessed by immunostaining for phosphorylated H2AX ( f , γH2AX, green, arrowheads). Arrowheads indicate γH2AX-positive cardiomyocytes and arrows indicate γH2AX-positive non-cardiomyocytes. Scale bar, 50 μm. The number of γH2AX-positive cardiomyocytes was counted ( g , n =4 each). Expression levels of inflammatory cytokines in the isolated cardiomyocytes were assessed by real-time PCR ( h , n =10, 16, 12 for each genotype, respectively, technical duplicates). ChIP–qPCR analysis of binding of NF-κB to the Vcam1 promoter region. Data is presented as fold enrichment relative to TAC-operated Xrcc1 f/f mice ( i , n =4, 5, 5, respectively). Heart tissues were immunostained for CD45 or CD68 ( j , green, arrowheads). Arrowheads indicate CD45- or CD68-positive cells. Scale bar, 50 μm. The number of CD45- and CD68-positive cells was counted ( k , n =4 each). Statistical significance was determined by one-way analysis of variance followed by the Tukey–Kramer HSD test for ( b ) (at each time point), ( c , g , h , i , k ), by Wilcoxon test for d and by Steel–Dwass test for e , # P
    Figure Legend Snippet: ATM gene deletion rescues the cardiac phenotypes of Xrcc1 deficient mice. ( a , b ) Macroscopic and echocardiographic images ( a ) and cardiac function ( b ) of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice ( Xrcc1 f/f mice: n =83, 21, 46, 11, 27; Xrcc1 αMHC-Cre mice: n =88, 28, 60, 13, 16; Xrcc1 αMHC-Cre ; Atm +/− mice: n =28, 22, 22, 7, 7 at each time point, respectively). Scale bar, 2 mm. ( c ) Heart, lung, and body weight of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice were weighed 8 weeks after the surgery ( n =8, 5, 6 for each genotype, respectively). ( d ) Survival curves of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice ( n =49, 62, 23, respectively). ( e – k ) TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice were analysed 4 weeks after the surgery. The type of DNA damage in cardiomyocytes was assessed by comet assay ( e , Alkaline comet: n =50, 76, 77; Neutral comet: n =53, 56, 42, respectively). Activation of DDR was assessed by immunostaining for phosphorylated H2AX ( f , γH2AX, green, arrowheads). Arrowheads indicate γH2AX-positive cardiomyocytes and arrows indicate γH2AX-positive non-cardiomyocytes. Scale bar, 50 μm. The number of γH2AX-positive cardiomyocytes was counted ( g , n =4 each). Expression levels of inflammatory cytokines in the isolated cardiomyocytes were assessed by real-time PCR ( h , n =10, 16, 12 for each genotype, respectively, technical duplicates). ChIP–qPCR analysis of binding of NF-κB to the Vcam1 promoter region. Data is presented as fold enrichment relative to TAC-operated Xrcc1 f/f mice ( i , n =4, 5, 5, respectively). Heart tissues were immunostained for CD45 or CD68 ( j , green, arrowheads). Arrowheads indicate CD45- or CD68-positive cells. Scale bar, 50 μm. The number of CD45- and CD68-positive cells was counted ( k , n =4 each). Statistical significance was determined by one-way analysis of variance followed by the Tukey–Kramer HSD test for ( b ) (at each time point), ( c , g , h , i , k ), by Wilcoxon test for d and by Steel–Dwass test for e , # P

    Techniques Used: Mouse Assay, Single Cell Gel Electrophoresis, Activation Assay, Immunostaining, Expressing, Isolation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay

    10) Product Images from "Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency"

    Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.06.002

    The LINC00116 -Derived Microprotein Mitoregulin Localizes to Inner Mitochondrial Membranes and Binds Cardiolipin (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.
    Figure Legend Snippet: The LINC00116 -Derived Microprotein Mitoregulin Localizes to Inner Mitochondrial Membranes and Binds Cardiolipin (A and B) Wild-type (A) and GFP-tagged (B) human Mtln were expressed in neonatal rat cardiomyocytes, and co-localization with MitoTracker red was evaluated. Representative photomicrographs are shown. Scale bars, 10 μm. (C) Mitochondrial pellets were isolated from wild-type (WT) or Mtln-knockout (KO) C2C12 myoblast cells, and western blot was performed on various fractions. (D) Mitochondrial pellets harvested from WT or Mtln-KO skeletal muscle tissues were treated with increasing digitonin concentrations to release OMMs, and pellet and supernatant fraction fractions were subjected to western blot analysis. Cox4 and Vdac1 are known IMM and OMM proteins, respectively. Gapdh is a cytosolic protein known to associate with mitochondria in some cases. (E) Mitochondrial pellets harvested from WT skeletal muscle tissues were resuspended in isotonic, hypotonic, or isotonic plus triton buffers in the absence or presence of proteinase K and subjected to western blot analysis. Proteins with known localization to various mitochondrial compartments (e.g., matrix, IMM, and intermembrane space [IMS]) were evaluated as controls. (F) Western blot analysis performed on WT and Mtln-KO cardiac tissue lysates subjected to pull-down assay using cardiolipin (CL)-coated or control beads. Subunit c, a known cardiolipin-binding protein, serves as the positive control. (G) Lipid-strip binding assay performed using synthetic Mtln protein followed by anti-Mtln immunoblot.

    Techniques Used: Derivative Assay, Isolation, Knock-Out, Western Blot, Pull Down Assay, Binding Assay, Positive Control, Stripping Membranes

    11) Product Images from "DNA single-strand break-induced DNA damage response causes heart failure"

    Article Title: DNA single-strand break-induced DNA damage response causes heart failure

    Journal: Nature Communications

    doi: 10.1038/ncomms15104

    Generation of an in vitro model of cardiomyocytes with SSB accumulation. ( a ) Neonatal rat cardiomyocytes (NRCMs) were treated with MMS at the indicated concentration for 10 min and the DNA damage was analysed by comet assay (Alkaline comet: n =42, 37, 45, 33, 34; Neutral comet: n =40, 35, 35, 37, 29 at each concentration, respectively). Statistical significance was determined by Steel–Dwass test. ## P
    Figure Legend Snippet: Generation of an in vitro model of cardiomyocytes with SSB accumulation. ( a ) Neonatal rat cardiomyocytes (NRCMs) were treated with MMS at the indicated concentration for 10 min and the DNA damage was analysed by comet assay (Alkaline comet: n =42, 37, 45, 33, 34; Neutral comet: n =40, 35, 35, 37, 29 at each concentration, respectively). Statistical significance was determined by Steel–Dwass test. ## P

    Techniques Used: In Vitro, Concentration Assay, Single Cell Gel Electrophoresis

    Possible roles of SSB accumulation in pathogenesis of heart failure. Accumulation of DNA SSB in cardiomyocytes induces persistent activation of DDR and subsequent activation of NF-κB pathway, resulting in increased expressions of inflammatory cytokines. These mechanisms may contribute, at least in part, to increased cardiac inflammation and the progression of pressure overload-induced heart failure.
    Figure Legend Snippet: Possible roles of SSB accumulation in pathogenesis of heart failure. Accumulation of DNA SSB in cardiomyocytes induces persistent activation of DDR and subsequent activation of NF-κB pathway, resulting in increased expressions of inflammatory cytokines. These mechanisms may contribute, at least in part, to increased cardiac inflammation and the progression of pressure overload-induced heart failure.

    Techniques Used: Activation Assay

    Xrcc1 deficiency exacerbates cardiac inflammation after pressure overload. ( a , b ) Activation of DDR in Sham- or TAC-operated Xrcc1 f/f and Xrcc1 αMHC-Cre mice was assessed by immunostaining for phosphorylated H2AX ( a , γH2AX, green). Immunostaining for alpha-actinin (red) was used to label cardiomyocytes. Arrowheads indicate γH2AX-positive cardiomyocytes and arrows indicate γH2AX-positive non-cardiomyocytes. Scale bar, 50 μm. The number of γH2AX-positive cardiomyocytes was counted ( b , n =4 each). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Tukey–Kramer HSD test. ** P
    Figure Legend Snippet: Xrcc1 deficiency exacerbates cardiac inflammation after pressure overload. ( a , b ) Activation of DDR in Sham- or TAC-operated Xrcc1 f/f and Xrcc1 αMHC-Cre mice was assessed by immunostaining for phosphorylated H2AX ( a , γH2AX, green). Immunostaining for alpha-actinin (red) was used to label cardiomyocytes. Arrowheads indicate γH2AX-positive cardiomyocytes and arrows indicate γH2AX-positive non-cardiomyocytes. Scale bar, 50 μm. The number of γH2AX-positive cardiomyocytes was counted ( b , n =4 each). Statistical significance was determined by one-way analysis of variance (ANOVA) followed by the Tukey–Kramer HSD test. ** P

    Techniques Used: Activation Assay, Mouse Assay, Immunostaining

    Accumulation of DNA SSB in the failing heart. ( a , b ) Cardiomyocytes were isolated from the TAC-operated heart at the indicated time points. The type of DNA damage in cardiomyocytes was assessed by comet assay. Representative images ( a ) and quantitative analyses are shown ( b , Alkaline comet: n =28, 45, 48; Neutral comet: n =38, 56, 44 at each time point, respectively, biological replicates=3). ( c , d ) Fragmented DNA and DSB were labelled with ISOL staining ( c , green). Wheat germ agglutinin (WGA, red) was used to visualize cardiomyocytes. DNase-treated section (DNase I, 10 Kunitz units ml −1 ) was used as a positive control. Arrowheads indicate ISOL-positive cardiomyocytes and arrows indicate ISOL-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. The number of ISOL-positive cardiomyocytes was counted ( d , n =4 each). ( e , f ) Heart tissue sections were immunostained for NBS1 ( e , NBS1, green). Immunostaining for alpha-actinin (red) was used to label cardiomyocytes. Scale bar, 50 μm. The number of NBS1-positive cardiomyocytes was counted ( f , n =4 each). ( g , h ) Heart tissue sections were immunostained for poly-ADP ribose ( g , PAR, green) and the number of PAR-positive cardiomyocytes was counted ( h , n =4, 4, 5 at each time point, respectively). Arrowheads indicate PAR-positive cardiomyocytes and arrows indicate PAR-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. ( i ) Expression levels of SSB repair enzymes were analysed by real-time PCR ( n =4, 6, 8 at each time point, respectively, technical duplicates). ( j , k ) Heart tissue sections were stained with dihydroethidium ( i , DHE, 10 μΜ) and mean fluorescence intensity relative to Sham-operated mice was measured ( k , n =4, 5, 5 at each time point, respectively). Scale bar, 50 μm. ( l ) The level of H 2 O 2 in the TAC-operated heart was measured using Amplex Red assay ( n =9, 5, 6 at each time point, respectively). Statistical significance was determined by Steel-Dwass test for ( b ) and by one-way analysis of variance followed by the Tukey–Kramer HSD test for ( d , f , h , i , j ) * P
    Figure Legend Snippet: Accumulation of DNA SSB in the failing heart. ( a , b ) Cardiomyocytes were isolated from the TAC-operated heart at the indicated time points. The type of DNA damage in cardiomyocytes was assessed by comet assay. Representative images ( a ) and quantitative analyses are shown ( b , Alkaline comet: n =28, 45, 48; Neutral comet: n =38, 56, 44 at each time point, respectively, biological replicates=3). ( c , d ) Fragmented DNA and DSB were labelled with ISOL staining ( c , green). Wheat germ agglutinin (WGA, red) was used to visualize cardiomyocytes. DNase-treated section (DNase I, 10 Kunitz units ml −1 ) was used as a positive control. Arrowheads indicate ISOL-positive cardiomyocytes and arrows indicate ISOL-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. The number of ISOL-positive cardiomyocytes was counted ( d , n =4 each). ( e , f ) Heart tissue sections were immunostained for NBS1 ( e , NBS1, green). Immunostaining for alpha-actinin (red) was used to label cardiomyocytes. Scale bar, 50 μm. The number of NBS1-positive cardiomyocytes was counted ( f , n =4 each). ( g , h ) Heart tissue sections were immunostained for poly-ADP ribose ( g , PAR, green) and the number of PAR-positive cardiomyocytes was counted ( h , n =4, 4, 5 at each time point, respectively). Arrowheads indicate PAR-positive cardiomyocytes and arrows indicate PAR-positive non-cardiomyocytes. White scale bar, 50 μm; yellow scale bar, 20 μm. ( i ) Expression levels of SSB repair enzymes were analysed by real-time PCR ( n =4, 6, 8 at each time point, respectively, technical duplicates). ( j , k ) Heart tissue sections were stained with dihydroethidium ( i , DHE, 10 μΜ) and mean fluorescence intensity relative to Sham-operated mice was measured ( k , n =4, 5, 5 at each time point, respectively). Scale bar, 50 μm. ( l ) The level of H 2 O 2 in the TAC-operated heart was measured using Amplex Red assay ( n =9, 5, 6 at each time point, respectively). Statistical significance was determined by Steel-Dwass test for ( b ) and by one-way analysis of variance followed by the Tukey–Kramer HSD test for ( d , f , h , i , j ) * P

    Techniques Used: Isolation, Single Cell Gel Electrophoresis, Staining, Whole Genome Amplification, Positive Control, Immunostaining, Expressing, Real-time Polymerase Chain Reaction, Fluorescence, Mouse Assay, Amplex Red Assay

    ATM gene deletion rescues the cardiac phenotypes of Xrcc1 deficient mice. ( a , b ) Macroscopic and echocardiographic images ( a ) and cardiac function ( b ) of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice ( Xrcc1 f/f mice: n =83, 21, 46, 11, 27; Xrcc1 αMHC-Cre mice: n =88, 28, 60, 13, 16; Xrcc1 αMHC-Cre ; Atm +/− mice: n =28, 22, 22, 7, 7 at each time point, respectively). Scale bar, 2 mm. ( c ) Heart, lung, and body weight of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice were weighed 8 weeks after the surgery ( n =8, 5, 6 for each genotype, respectively). ( d ) Survival curves of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice ( n =49, 62, 23, respectively). ( e – k ) TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice were analysed 4 weeks after the surgery. The type of DNA damage in cardiomyocytes was assessed by comet assay ( e , Alkaline comet: n =50, 76, 77; Neutral comet: n =53, 56, 42, respectively). Activation of DDR was assessed by immunostaining for phosphorylated H2AX ( f , γH2AX, green, arrowheads). Arrowheads indicate γH2AX-positive cardiomyocytes and arrows indicate γH2AX-positive non-cardiomyocytes. Scale bar, 50 μm. The number of γH2AX-positive cardiomyocytes was counted ( g , n =4 each). Expression levels of inflammatory cytokines in the isolated cardiomyocytes were assessed by real-time PCR ( h , n =10, 16, 12 for each genotype, respectively, technical duplicates). ChIP–qPCR analysis of binding of NF-κB to the Vcam1 promoter region. Data is presented as fold enrichment relative to TAC-operated Xrcc1 f/f mice ( i , n =4, 5, 5, respectively). Heart tissues were immunostained for CD45 or CD68 ( j , green, arrowheads). Arrowheads indicate CD45- or CD68-positive cells. Scale bar, 50 μm. The number of CD45- and CD68-positive cells was counted ( k , n =4 each). Statistical significance was determined by one-way analysis of variance followed by the Tukey–Kramer HSD test for ( b ) (at each time point), ( c , g , h , i , k ), by Wilcoxon test for d and by Steel–Dwass test for e , # P
    Figure Legend Snippet: ATM gene deletion rescues the cardiac phenotypes of Xrcc1 deficient mice. ( a , b ) Macroscopic and echocardiographic images ( a ) and cardiac function ( b ) of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice ( Xrcc1 f/f mice: n =83, 21, 46, 11, 27; Xrcc1 αMHC-Cre mice: n =88, 28, 60, 13, 16; Xrcc1 αMHC-Cre ; Atm +/− mice: n =28, 22, 22, 7, 7 at each time point, respectively). Scale bar, 2 mm. ( c ) Heart, lung, and body weight of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice were weighed 8 weeks after the surgery ( n =8, 5, 6 for each genotype, respectively). ( d ) Survival curves of TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice ( n =49, 62, 23, respectively). ( e – k ) TAC-operated Xrcc1 f/f , Xrcc1 αMHC-Cre and Xrcc1 αMHC-Cre ; Atm +/− mice were analysed 4 weeks after the surgery. The type of DNA damage in cardiomyocytes was assessed by comet assay ( e , Alkaline comet: n =50, 76, 77; Neutral comet: n =53, 56, 42, respectively). Activation of DDR was assessed by immunostaining for phosphorylated H2AX ( f , γH2AX, green, arrowheads). Arrowheads indicate γH2AX-positive cardiomyocytes and arrows indicate γH2AX-positive non-cardiomyocytes. Scale bar, 50 μm. The number of γH2AX-positive cardiomyocytes was counted ( g , n =4 each). Expression levels of inflammatory cytokines in the isolated cardiomyocytes were assessed by real-time PCR ( h , n =10, 16, 12 for each genotype, respectively, technical duplicates). ChIP–qPCR analysis of binding of NF-κB to the Vcam1 promoter region. Data is presented as fold enrichment relative to TAC-operated Xrcc1 f/f mice ( i , n =4, 5, 5, respectively). Heart tissues were immunostained for CD45 or CD68 ( j , green, arrowheads). Arrowheads indicate CD45- or CD68-positive cells. Scale bar, 50 μm. The number of CD45- and CD68-positive cells was counted ( k , n =4 each). Statistical significance was determined by one-way analysis of variance followed by the Tukey–Kramer HSD test for ( b ) (at each time point), ( c , g , h , i , k ), by Wilcoxon test for d and by Steel–Dwass test for e , # P

    Techniques Used: Mouse Assay, Single Cell Gel Electrophoresis, Activation Assay, Immunostaining, Expressing, Isolation, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Binding Assay

    12) Product Images from "BAG3 Directly Interacts with Mutated alphaB-Crystallin to Suppress Its Aggregation and Toxicity"

    Article Title: BAG3 Directly Interacts with Mutated alphaB-Crystallin to Suppress Its Aggregation and Toxicity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016828

    BAG3 binds αB-crystallin directly through BAG3 intermediate region. (A) BAG3 interacts with αB-crystallin in rat cardiomyocytes . Cardiomyocytes were isolated from rat hearts, and Flag-tagged BAG3 was expressed using adenovirus. Cell extracts were immunoprecipitated with anti-Flag antibody, and the sample was applied to SDS-PAGE. Anti-αB-crystallin antibody was used to detect αB-crystallin. The same membrane was blotted with anti-Hsc70 and anti-αActinin antibody for a positive and negative control, respectively. No actinin interacting was observed (negative control). (B) Schematic representation of the primary structure of wild-type BAG3 and its mutants . The BAG3ΔC construct lacks the BAG domain. The BAG3 intermediate domain and amino acids from 87–101 and from 200 to 213 are deleted in BAG3ΔM1 and BAG3ΔM2, respectively. Amino acid sequences between residues 87–101 and 200–213 are shown. (C) Co-precipitation of αB-crystallin and BAG3 . Flag-tagged BAG3 or mutants were expressed with αB-crystallin in HEK293 cells, and cell extracts were immunoprecipitated with anti-Flag antibody. αB-crystallin was detected with anti-αB-crystallin antibody. Anti-Hsc70 antibody was used to detect Hsc70 precipitation with BAG3. The cell extracts were also applied to an immunoblotting assay to verify expression. (D) BAG3 interacts with αB-crystallin via amino acids 87–101 and 200–213 . His-tagged BAG3 or BAG3M2 was expressed with αB-crystallin in HEK293 cells, and BAG3 was precipitated with anti-His antibody. Co-precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane was blotted with anti-His antibody (bottom panel). (E) Direct interaction of BAG3 with αB-crystallin . Left panel: GST protein fused to BAG3 was expressed in Escherichia coli , and purified fusion proteins were incubated with purified αB-crystallin, followed by precipitation with GSH beads. After SDS-PAGE, αB-crystallin was detected using anti-αB-crystallin antibody. Right panel: GST fusion proteins used in the experiment were visualized with CBB staining to verify the quality and quantity. (F) Competition assay with peptides . Purified GST or GST-BAG3 was incubated with purified αB-crystallin in the presence or absence of peptides corresponding to amino acids 87–101 or 200–213 of BAG3 at indicated concentrations. The precipitated samples with GSH beads were loaded onto SDS-PAGE, followed by an immunoblotting assay using anti-αB-crystallin antibody.
    Figure Legend Snippet: BAG3 binds αB-crystallin directly through BAG3 intermediate region. (A) BAG3 interacts with αB-crystallin in rat cardiomyocytes . Cardiomyocytes were isolated from rat hearts, and Flag-tagged BAG3 was expressed using adenovirus. Cell extracts were immunoprecipitated with anti-Flag antibody, and the sample was applied to SDS-PAGE. Anti-αB-crystallin antibody was used to detect αB-crystallin. The same membrane was blotted with anti-Hsc70 and anti-αActinin antibody for a positive and negative control, respectively. No actinin interacting was observed (negative control). (B) Schematic representation of the primary structure of wild-type BAG3 and its mutants . The BAG3ΔC construct lacks the BAG domain. The BAG3 intermediate domain and amino acids from 87–101 and from 200 to 213 are deleted in BAG3ΔM1 and BAG3ΔM2, respectively. Amino acid sequences between residues 87–101 and 200–213 are shown. (C) Co-precipitation of αB-crystallin and BAG3 . Flag-tagged BAG3 or mutants were expressed with αB-crystallin in HEK293 cells, and cell extracts were immunoprecipitated with anti-Flag antibody. αB-crystallin was detected with anti-αB-crystallin antibody. Anti-Hsc70 antibody was used to detect Hsc70 precipitation with BAG3. The cell extracts were also applied to an immunoblotting assay to verify expression. (D) BAG3 interacts with αB-crystallin via amino acids 87–101 and 200–213 . His-tagged BAG3 or BAG3M2 was expressed with αB-crystallin in HEK293 cells, and BAG3 was precipitated with anti-His antibody. Co-precipitated αB-crystallin was detected with anti-αB-crystallin antibody (upper panel). The same membrane was blotted with anti-His antibody (bottom panel). (E) Direct interaction of BAG3 with αB-crystallin . Left panel: GST protein fused to BAG3 was expressed in Escherichia coli , and purified fusion proteins were incubated with purified αB-crystallin, followed by precipitation with GSH beads. After SDS-PAGE, αB-crystallin was detected using anti-αB-crystallin antibody. Right panel: GST fusion proteins used in the experiment were visualized with CBB staining to verify the quality and quantity. (F) Competition assay with peptides . Purified GST or GST-BAG3 was incubated with purified αB-crystallin in the presence or absence of peptides corresponding to amino acids 87–101 or 200–213 of BAG3 at indicated concentrations. The precipitated samples with GSH beads were loaded onto SDS-PAGE, followed by an immunoblotting assay using anti-αB-crystallin antibody.

    Techniques Used: Isolation, Immunoprecipitation, SDS Page, Negative Control, Construct, Expressing, Purification, Incubation, Staining, Competitive Binding Assay

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    Expressing:

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