cardiomyocyte medium  (Thermo Fisher)


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    Structured Review

    Thermo Fisher cardiomyocyte medium
    Chronic 3HB exposure induces CD36-mediated lipid accumulation in <t>cardiomyocytes.</t> ARCMs were cultured for 24 h in either low palmitate <t>medium</t> (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB), or LP with 3HB in the presence of 4*KLR (3HB/4*KLR). ( A,B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. ( A ) Assessment of cell-surface CD36 using biotinylation assay. For this, CD36 was detected by Western blotting in biotin immunoprecipitations and total cell lysates and subsequently quantified (n = 6). ( B ) [14C]palmitate uptake (n = 6). ( C ) Triacylglycerol contents (n = 7). Bar values are means ± SEM. * p
    Cardiomyocyte Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly—Rescue by Specific Amino Acid Supplementation"

    Article Title: Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly—Rescue by Specific Amino Acid Supplementation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms232112909

    Chronic 3HB exposure induces CD36-mediated lipid accumulation in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB), or LP with 3HB in the presence of 4*KLR (3HB/4*KLR). ( A,B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. ( A ) Assessment of cell-surface CD36 using biotinylation assay. For this, CD36 was detected by Western blotting in biotin immunoprecipitations and total cell lysates and subsequently quantified (n = 6). ( B ) [14C]palmitate uptake (n = 6). ( C ) Triacylglycerol contents (n = 7). Bar values are means ± SEM. * p
    Figure Legend Snippet: Chronic 3HB exposure induces CD36-mediated lipid accumulation in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB), or LP with 3HB in the presence of 4*KLR (3HB/4*KLR). ( A,B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. ( A ) Assessment of cell-surface CD36 using biotinylation assay. For this, CD36 was detected by Western blotting in biotin immunoprecipitations and total cell lysates and subsequently quantified (n = 6). ( B ) [14C]palmitate uptake (n = 6). ( C ) Triacylglycerol contents (n = 7). Bar values are means ± SEM. * p

    Techniques Used: Cell Culture, Incubation, Cell Surface Biotinylation Assay, Western Blot

    Chronic 3HB exposure prevents insulin-stimulated GLUT4 translocation and glucose uptake in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB); LP with 3HB in the presence of 4*KLR (3HB/4*KLR); or LP with 3 mM acetoacetate (AcAc). ( A , B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. ( A ) Microscopical assay of cell-surface GLUT4. aRCMs were transduced for 24 h with an adenoviral vector containing an HA-GLUT4-GFP fusion protein before the start of the culturing. Non-permeabilized cells were anti-HA immunostained. The nuclei are stained in blue (DAPI). Representative microscopical images are displayed (the scale bar is 50 µm). The ratios of red (HA-tag) and green (GFP) intensity per pixel were quantified by Image J (n = 6). ( B ) Biotinylation assay. Representative Western blot and quantification of insulin-regulated aminopeptidase (IRAP, which reflects GLUT4 translocation) in biotin-immunoprecipitations and in total lysates (n = 6). ( C ) [3H]Deoxyglucose uptake (n ≥ 6). Bar values are means ± SEM. * p
    Figure Legend Snippet: Chronic 3HB exposure prevents insulin-stimulated GLUT4 translocation and glucose uptake in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB); LP with 3HB in the presence of 4*KLR (3HB/4*KLR); or LP with 3 mM acetoacetate (AcAc). ( A , B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. ( A ) Microscopical assay of cell-surface GLUT4. aRCMs were transduced for 24 h with an adenoviral vector containing an HA-GLUT4-GFP fusion protein before the start of the culturing. Non-permeabilized cells were anti-HA immunostained. The nuclei are stained in blue (DAPI). Representative microscopical images are displayed (the scale bar is 50 µm). The ratios of red (HA-tag) and green (GFP) intensity per pixel were quantified by Image J (n = 6). ( B ) Biotinylation assay. Representative Western blot and quantification of insulin-regulated aminopeptidase (IRAP, which reflects GLUT4 translocation) in biotin-immunoprecipitations and in total lysates (n = 6). ( C ) [3H]Deoxyglucose uptake (n ≥ 6). Bar values are means ± SEM. * p

    Techniques Used: Translocation Assay, Cell Culture, Incubation, Plasmid Preparation, Staining, Cell Surface Biotinylation Assay, Western Blot

    Chronic 3HB exposure reduces v-ATPase activity and insulin sensitivity in human cardiomyocytes. HiPSC-CMs were cultured for 24 h in either control medium (Ctrl), high palmitate medium (HP), Ctrl with 3mM Acetoacetate (AcAc), Ctrl with 3mM 3-β-hydroxybutyrate (3HB), 3HB with supplementation of the 4*KLR mixture (3HB/4*KLR), and with 100 nM Baf (3HB/4*KLR/BafA). ( A ) V-ATPase function in hiPSC-CMs: after culturing, cells were subjected to the [3H] CHLQ accumulation assay (n = 9). ( B , C ) After 24 h, cells were short-term (30 min) incubated without/with 200 nM insulin. ( B ) [14C]palmitate uptake (n = 6). ( C ) [3H] deoxyglucose uptake (n = 3). ( D ) Insulin signaling: Western analysis of phosphorylation of Akt and S6 at Ser473 and Ser235/236, respectively. Representative blots of pAKT and pS6 and Caveolin-3 (loading control) are displayed (n = 4). Bar values are means ± SEM. * p
    Figure Legend Snippet: Chronic 3HB exposure reduces v-ATPase activity and insulin sensitivity in human cardiomyocytes. HiPSC-CMs were cultured for 24 h in either control medium (Ctrl), high palmitate medium (HP), Ctrl with 3mM Acetoacetate (AcAc), Ctrl with 3mM 3-β-hydroxybutyrate (3HB), 3HB with supplementation of the 4*KLR mixture (3HB/4*KLR), and with 100 nM Baf (3HB/4*KLR/BafA). ( A ) V-ATPase function in hiPSC-CMs: after culturing, cells were subjected to the [3H] CHLQ accumulation assay (n = 9). ( B , C ) After 24 h, cells were short-term (30 min) incubated without/with 200 nM insulin. ( B ) [14C]palmitate uptake (n = 6). ( C ) [3H] deoxyglucose uptake (n = 3). ( D ) Insulin signaling: Western analysis of phosphorylation of Akt and S6 at Ser473 and Ser235/236, respectively. Representative blots of pAKT and pS6 and Caveolin-3 (loading control) are displayed (n = 4). Bar values are means ± SEM. * p

    Techniques Used: Activity Assay, Cell Culture, Incubation, Western Blot

    Chronic 3HB exposure causes v-ATPase disassembly and de-activation in cardiomyocytes. ( A ) 3HB exposure results in the loss of v-ATPase activity (measured as [3H]chloroquine accumulation) in cardiomyocytes: aRCMs were cultured for 24 h in either low palmitate medium (LP; control condition); LP with 100 nM bafilomycin-A (BafA), LP with 3 mM acetoacetate (AcAc); LP with different concentrations of 3-β-hydroxybutyrate (3HB; 1mM, 3 mM, and 9 mM), high palmitate medium (HP), HP with 3mM AcAc (HP/AcAc), or HP with 3mM 3HB (HP/3HB). Directly after the culturing, cells were used for the [3H]chloroquine accumulation assay. (n = 5). ( B ) Amino acids prevent 3HB-induced v-ATPase inhibition in cardiomyocytes: aRCMs were incubated for 24 h in either LP, HP, BafA, LP supplemented with 1.56 mM Lys, 1.84 mM Leu and 1.36 mM Arg, (LP/4*KLR; for explanation see Section 4.2 ), 3HB (3 mM), 3HB supplemented with the 4*KLR mix (3HB/4*KLR). Directly after the culturing, cells were used for the [3H]chloroquine accumulation assay (n = 5). ( C – F ) 3HB exposure induces v-ATPase disassembly: HL-1 cells were incubated for 24 h in either control (Ctrl) medium, HP medium, or Ctrl medium with 3 mM 3HB (3HB). ( C ) Subcellular fractionation: cytoplasmic fractions (C) and membrane fractions (M) were analyzed by Western blotting of v-ATPase subunits B2 (V1-B2) and d1 (V0-d1), after which the signals were quantified. For V1-B2, the signal ratio of membrane fraction/cytoplasm in control condition is set at 1.0. For V0-d1, the signal density in the membrane fraction is 1.0. The quantified signals in the other conditions are expressed as multiples. Representative blots are displayed. Caveolin-3 (Cav-3) and GAPDH: loading controls for membrane and cytoplasmic fraction, respectively. (n = 4). ( D – F ) Co-Immunoprecipitation (Co-IP) of V1, V0, and mTORC1. ( D ) IP with V1-B2, ( E ) IP with V0-d1. ( F ) IP with mTORC1. Immunoprecipitates were blotted with antibodies against mTORC1, V0-a2, V0-d1, and V1-B2, after which the signals were quantified. Representative Western blots are displayed. (n = 3). Bar values are means ± SEM. * p
    Figure Legend Snippet: Chronic 3HB exposure causes v-ATPase disassembly and de-activation in cardiomyocytes. ( A ) 3HB exposure results in the loss of v-ATPase activity (measured as [3H]chloroquine accumulation) in cardiomyocytes: aRCMs were cultured for 24 h in either low palmitate medium (LP; control condition); LP with 100 nM bafilomycin-A (BafA), LP with 3 mM acetoacetate (AcAc); LP with different concentrations of 3-β-hydroxybutyrate (3HB; 1mM, 3 mM, and 9 mM), high palmitate medium (HP), HP with 3mM AcAc (HP/AcAc), or HP with 3mM 3HB (HP/3HB). Directly after the culturing, cells were used for the [3H]chloroquine accumulation assay. (n = 5). ( B ) Amino acids prevent 3HB-induced v-ATPase inhibition in cardiomyocytes: aRCMs were incubated for 24 h in either LP, HP, BafA, LP supplemented with 1.56 mM Lys, 1.84 mM Leu and 1.36 mM Arg, (LP/4*KLR; for explanation see Section 4.2 ), 3HB (3 mM), 3HB supplemented with the 4*KLR mix (3HB/4*KLR). Directly after the culturing, cells were used for the [3H]chloroquine accumulation assay (n = 5). ( C – F ) 3HB exposure induces v-ATPase disassembly: HL-1 cells were incubated for 24 h in either control (Ctrl) medium, HP medium, or Ctrl medium with 3 mM 3HB (3HB). ( C ) Subcellular fractionation: cytoplasmic fractions (C) and membrane fractions (M) were analyzed by Western blotting of v-ATPase subunits B2 (V1-B2) and d1 (V0-d1), after which the signals were quantified. For V1-B2, the signal ratio of membrane fraction/cytoplasm in control condition is set at 1.0. For V0-d1, the signal density in the membrane fraction is 1.0. The quantified signals in the other conditions are expressed as multiples. Representative blots are displayed. Caveolin-3 (Cav-3) and GAPDH: loading controls for membrane and cytoplasmic fraction, respectively. (n = 4). ( D – F ) Co-Immunoprecipitation (Co-IP) of V1, V0, and mTORC1. ( D ) IP with V1-B2, ( E ) IP with V0-d1. ( F ) IP with mTORC1. Immunoprecipitates were blotted with antibodies against mTORC1, V0-a2, V0-d1, and V1-B2, after which the signals were quantified. Representative Western blots are displayed. (n = 3). Bar values are means ± SEM. * p

    Techniques Used: Activation Assay, Activity Assay, Cell Culture, Inhibition, Incubation, Fractionation, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

    Chronic 3HB exposure induces contractile dysfunction in cardiomyocytes. ARCMs were cultured for 24 h under various conditions, being low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB) or 3 mM acetoacetate (AcAc), and LP with 3HB in the presence of 4*KLR (3HB/4*KLR). The contractile parameters ( A ) sarcomere shortening, ( B ) time to peak and ( C ) decay time were deduced during electrostimulation at 1 Hz frequency (n = 4; imaging of 10 cells/measurement condition). Bar values are means ± SEM. * p
    Figure Legend Snippet: Chronic 3HB exposure induces contractile dysfunction in cardiomyocytes. ARCMs were cultured for 24 h under various conditions, being low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB) or 3 mM acetoacetate (AcAc), and LP with 3HB in the presence of 4*KLR (3HB/4*KLR). The contractile parameters ( A ) sarcomere shortening, ( B ) time to peak and ( C ) decay time were deduced during electrostimulation at 1 Hz frequency (n = 4; imaging of 10 cells/measurement condition). Bar values are means ± SEM. * p

    Techniques Used: Cell Culture, Imaging

    Chronic 3HB exposure interferes with insulin signaling in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB), or LP with 3HB in the presence of 4*KLR (3HB/4*KLR). ( A , B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. Thereafter, ( A ) Ser473 phosphorylation of Akt, ( B ) Ser2447 phosphorylation of mTOR, ( C ) Thr 642 phosphorylation of AS160, and ( D ) Ser235/236 phosphorylation of S6 were assessed by Western blotting and quantified. For this, normalization was performed against loading controls. Loading control for the degree of phosphorylation of Akt and AS160 is caveolin-3. Loading control for mTOR and S6 phosphorylation is GAPDHs. Representative blots and corresponding loading controls are displayed (n = 8). Bar values are means ± SEM. * p
    Figure Legend Snippet: Chronic 3HB exposure interferes with insulin signaling in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB), or LP with 3HB in the presence of 4*KLR (3HB/4*KLR). ( A , B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. Thereafter, ( A ) Ser473 phosphorylation of Akt, ( B ) Ser2447 phosphorylation of mTOR, ( C ) Thr 642 phosphorylation of AS160, and ( D ) Ser235/236 phosphorylation of S6 were assessed by Western blotting and quantified. For this, normalization was performed against loading controls. Loading control for the degree of phosphorylation of Akt and AS160 is caveolin-3. Loading control for mTOR and S6 phosphorylation is GAPDHs. Representative blots and corresponding loading controls are displayed (n = 8). Bar values are means ± SEM. * p

    Techniques Used: Cell Culture, Incubation, Western Blot

    2) Product Images from "Circulating cardiomyocyte-derived extracellular vesicles reflect cardiac injury during systemic inflammatory response syndrome in mice"

    Article Title: Circulating cardiomyocyte-derived extracellular vesicles reflect cardiac injury during systemic inflammatory response syndrome in mice

    Journal: Cellular and Molecular Life Sciences

    doi: 10.1007/s00018-021-04125-w

    Tissue-specific and inducible Cre-mediated expression of GFP in cardiomyocytes. a Generation of tamoxifen-inducible double transgenic animals (MerCreMer/mTmG). Schematic illustration of MerCreMer and mTmG construct to generate bi-transgenic animals. MerCreMer mice are crossed with mTmG animals to trace the lineage of GFP-positive cells in the cardiomyocyte. b Ubiquitous expression of tdTomato and cardiomyocyte-specific expression of GFP demonstrated in mouse tissue from a MerCreMer x mTmG cross at necropsy. Fluorescent, F1 generation littermates were euthanized and imaged at 12 weeks of age. All sections were recorded during a single imaging session using the same exposure times. GFP expression is limited to the cardiomyocyte whereas tdTomato is brightly expressed in all the other cells. The images of different muscle tissues were adjusted equally for brightness and contrast. All other images are shown without post-acquisition processing. Scale bars, 100 μm. c Cardiomyocytes were isolated from adult transgenic mice. The left panel shows the isolated GFP expressing cardiomyocytes under the 20X objective, the right panel shows the bright field image of the isolated single cells. Scale bars: 30 µm
    Figure Legend Snippet: Tissue-specific and inducible Cre-mediated expression of GFP in cardiomyocytes. a Generation of tamoxifen-inducible double transgenic animals (MerCreMer/mTmG). Schematic illustration of MerCreMer and mTmG construct to generate bi-transgenic animals. MerCreMer mice are crossed with mTmG animals to trace the lineage of GFP-positive cells in the cardiomyocyte. b Ubiquitous expression of tdTomato and cardiomyocyte-specific expression of GFP demonstrated in mouse tissue from a MerCreMer x mTmG cross at necropsy. Fluorescent, F1 generation littermates were euthanized and imaged at 12 weeks of age. All sections were recorded during a single imaging session using the same exposure times. GFP expression is limited to the cardiomyocyte whereas tdTomato is brightly expressed in all the other cells. The images of different muscle tissues were adjusted equally for brightness and contrast. All other images are shown without post-acquisition processing. Scale bars, 100 μm. c Cardiomyocytes were isolated from adult transgenic mice. The left panel shows the isolated GFP expressing cardiomyocytes under the 20X objective, the right panel shows the bright field image of the isolated single cells. Scale bars: 30 µm

    Techniques Used: Expressing, Transgenic Assay, Construct, Mouse Assay, Imaging, Isolation

    Ex vivo release of mEVs by primary adult murine cardiomyocytes upon in vivo administration of LPS. a Representative results of size distribution analysis (NTA) of mEVs from isolated cardiomyocytes from LPS-injected and control mice (24 h conditioned medium). b mEVs release was significantly elevated after 24 h. Data are obtained from three independent experiments (mean ± SD values are indicated in the figure) (* P
    Figure Legend Snippet: Ex vivo release of mEVs by primary adult murine cardiomyocytes upon in vivo administration of LPS. a Representative results of size distribution analysis (NTA) of mEVs from isolated cardiomyocytes from LPS-injected and control mice (24 h conditioned medium). b mEVs release was significantly elevated after 24 h. Data are obtained from three independent experiments (mean ± SD values are indicated in the figure) (* P

    Techniques Used: Ex Vivo, In Vivo, Isolation, Injection, Mouse Assay

    Effect of LPS administration on GFP + EV release to the circulation. a Schematic protocol of Tamoxifen induction and LPS injection of mice. b , c Flow cytometry analysis. Calibration beads were used (0.2–1 µm). d The gating strategy for mEVs and the 1 μm counting beads. e As a negative control, PFP-derived mEVs were analyzed from C57Bl6 mice. f Cardiomyocyte-derived EVs as GFP + events. mEVs secreted by all other cells were Tomato + . g A 95 ± 1% reduction of the event number after exposure to 0.1% Triton. h , i Events within the mEV gate were then analyzed, and GFP + events with/without LPS injection are shown on ( h ). i demonstrates Tomato + events with/without LPS injection. The figure shows the results of three independent experiments ( n = 9). The figure represents data from individual mEV samples (mice) as well the means ± SD (* P
    Figure Legend Snippet: Effect of LPS administration on GFP + EV release to the circulation. a Schematic protocol of Tamoxifen induction and LPS injection of mice. b , c Flow cytometry analysis. Calibration beads were used (0.2–1 µm). d The gating strategy for mEVs and the 1 μm counting beads. e As a negative control, PFP-derived mEVs were analyzed from C57Bl6 mice. f Cardiomyocyte-derived EVs as GFP + events. mEVs secreted by all other cells were Tomato + . g A 95 ± 1% reduction of the event number after exposure to 0.1% Triton. h , i Events within the mEV gate were then analyzed, and GFP + events with/without LPS injection are shown on ( h ). i demonstrates Tomato + events with/without LPS injection. The figure shows the results of three independent experiments ( n = 9). The figure represents data from individual mEV samples (mice) as well the means ± SD (* P

    Techniques Used: Injection, Mouse Assay, Flow Cytometry, Negative Control, Derivative Assay

    3) Product Images from "Alternative Splicing of Pericentrin Contributes to Cell Cycle Control in Cardiomyocytes"

    Article Title: Alternative Splicing of Pericentrin Contributes to Cell Cycle Control in Cardiomyocytes

    Journal: Journal of Cardiovascular Development and Disease

    doi: 10.3390/jcdd8080087

    Depletion of Pcnt S increases cell-cycle activity and DNA synthesis in postnatal cardiomyocytes. ( A , D ) Representative examples of P3 cardiomyocytes (troponin I) stained for Ki67 ( A ) and incorporated EdU ( D ). Nuclei were visualized with DAPI (DNA). ( B , E ) Quantitative analysis of Ki67 + ( B ) and EdU + ( E ) cardiomyocytes upon control (mock-transfected) and Pcnt isoform depletion as indicated. ( C , F ) Bayesian posterior distribution for the data in ( B , E ). Scale bars: 50 µm. White arrows: cardiomyocyte nuclei. Data are mean ± SD, **: p
    Figure Legend Snippet: Depletion of Pcnt S increases cell-cycle activity and DNA synthesis in postnatal cardiomyocytes. ( A , D ) Representative examples of P3 cardiomyocytes (troponin I) stained for Ki67 ( A ) and incorporated EdU ( D ). Nuclei were visualized with DAPI (DNA). ( B , E ) Quantitative analysis of Ki67 + ( B ) and EdU + ( E ) cardiomyocytes upon control (mock-transfected) and Pcnt isoform depletion as indicated. ( C , F ) Bayesian posterior distribution for the data in ( B , E ). Scale bars: 50 µm. White arrows: cardiomyocyte nuclei. Data are mean ± SD, **: p

    Techniques Used: Activity Assay, DNA Synthesis, Staining, Transfection

    Pcnt S depletion enhances serum-induced cell division in cardiomyocytes. ( A ) Quantitative analysis of all mitotic cardiomyocyte within 3 independent experiments upon control and Pcnt isoform depletion as indicated. For the experiments, ≥18,000 cardiomyocytes were analyzed per experimental condition. An object classifier was utilized that identifies mitotic cells based on DNA staining patterns. ( B ) Classification of the observed mitotic cardiomyocytes into pro(-meta)phase, metaphase, and ana-/telophase. ( C , E ) Quantification of binucleation ( C ) and mononucleated cardiomyocyte density ( E ). For the experiments ≥16,023 cardiomyocytes were analyzed per experimental condition. Data are mean ± SD, n = 3, n.s.: p > 0.05, *: p
    Figure Legend Snippet: Pcnt S depletion enhances serum-induced cell division in cardiomyocytes. ( A ) Quantitative analysis of all mitotic cardiomyocyte within 3 independent experiments upon control and Pcnt isoform depletion as indicated. For the experiments, ≥18,000 cardiomyocytes were analyzed per experimental condition. An object classifier was utilized that identifies mitotic cells based on DNA staining patterns. ( B ) Classification of the observed mitotic cardiomyocytes into pro(-meta)phase, metaphase, and ana-/telophase. ( C , E ) Quantification of binucleation ( C ) and mononucleated cardiomyocyte density ( E ). For the experiments ≥16,023 cardiomyocytes were analyzed per experimental condition. Data are mean ± SD, n = 3, n.s.: p > 0.05, *: p

    Techniques Used: Staining

    Immunofluorescence analysis of Pcnt S and Pcnt B localization. P3 cardiomyocytes were mock-transfected (Control) or transfected with SiRNAs to deplete Pcnt S or Pcnt S + B as indicated. ( A ) Immunofluorescence analysis of Pcnt expression in cardiomyocytes (troponin I) utilizing an antibody binding to both Pcnt S and Pcnt B (Pcnt B + S). Nuclei were visualized with DAPI (DNA). ( B ) Semi-quantitative analysis of median intensity of nuclear Pcnt B + S signal in ( A ). ( C ) Immunofluorescence analysis of Pcnt expression in cardiomyocytes (troponin I) utilizing an antibody binding to both Pcnt S and Pcnt B (Pcnt B + S) and an antibody detecting specifically Pcnt B. Nuclei were visualized with DAPI (DNA). ( D ) Semi-quantitative analysis of maximum intensity of the Pcnt B signal per cardiomyocyte in ( C ) as an approximation of the centrosomal Pcnt B signal. Yellow asterisk: cardiomyocyte nucleus. White arrows: nuclear envelope of cardiomyocytes. For the experiments, ≥ 2000 cardiomyocytes were analyzed per experimental condition. Scale bars: 50 µm. Data are mean ± SD, n = 3, *: p
    Figure Legend Snippet: Immunofluorescence analysis of Pcnt S and Pcnt B localization. P3 cardiomyocytes were mock-transfected (Control) or transfected with SiRNAs to deplete Pcnt S or Pcnt S + B as indicated. ( A ) Immunofluorescence analysis of Pcnt expression in cardiomyocytes (troponin I) utilizing an antibody binding to both Pcnt S and Pcnt B (Pcnt B + S). Nuclei were visualized with DAPI (DNA). ( B ) Semi-quantitative analysis of median intensity of nuclear Pcnt B + S signal in ( A ). ( C ) Immunofluorescence analysis of Pcnt expression in cardiomyocytes (troponin I) utilizing an antibody binding to both Pcnt S and Pcnt B (Pcnt B + S) and an antibody detecting specifically Pcnt B. Nuclei were visualized with DAPI (DNA). ( D ) Semi-quantitative analysis of maximum intensity of the Pcnt B signal per cardiomyocyte in ( C ) as an approximation of the centrosomal Pcnt B signal. Yellow asterisk: cardiomyocyte nucleus. White arrows: nuclear envelope of cardiomyocytes. For the experiments, ≥ 2000 cardiomyocytes were analyzed per experimental condition. Scale bars: 50 µm. Data are mean ± SD, n = 3, *: p

    Techniques Used: Immunofluorescence, Transfection, Expressing, Binding Assay

    4) Product Images from "Alternative splicing of pericentrin contributes to cell cycle control in cardiomyocytes"

    Article Title: Alternative splicing of pericentrin contributes to cell cycle control in cardiomyocytes

    Journal: bioRxiv

    doi: 10.1101/2021.04.19.440474

    Localization of ectopically expressed Pcnt S and Pcnt B. FLAG-tagged Pcnt B (FLAG-Pcnt B) or Pcnt S (FLAG-Pcnt S) were expressed in P3 cardiomyocytes (troponin I) and their localization was assessed by staining for FLAG as well as centrioles/centrosome (γ-tubulin). Nuclei were visualized with DAPI (DNA). Orange arrows: centrioles/centrosomes. Scale bars: 10 μm.
    Figure Legend Snippet: Localization of ectopically expressed Pcnt S and Pcnt B. FLAG-tagged Pcnt B (FLAG-Pcnt B) or Pcnt S (FLAG-Pcnt S) were expressed in P3 cardiomyocytes (troponin I) and their localization was assessed by staining for FLAG as well as centrioles/centrosome (γ-tubulin). Nuclei were visualized with DAPI (DNA). Orange arrows: centrioles/centrosomes. Scale bars: 10 μm.

    Techniques Used: Staining

    Pcnt S depletion enhances serum-induced cell division in cardiomyocytes. ( A ) Quantitative analysis of all mitotic cardiomyocyte within 3 independent experiments upon control and Pcnt isoform depletion as indicated. For the experiments ≥ 18.000 cardiomyocytes were analyzed per experimental condition. An object classifier was utilized that identifies mitotic cells based on DNA staining patterns. ( B ) Classification of the observed mitotic cardiomyocytes into pro(-meta)phase, metaphase, and ana-/telophase. ( C , E ) Quantification of binucleation ( C ) and mononucleated cardiomyocyte density ( E ). For the experiments ≥ 16023 cardiomyocytes were analyzed per experimental condition. Data are mean ± SD, n = 3, n.s.: p > 0.05, *: p
    Figure Legend Snippet: Pcnt S depletion enhances serum-induced cell division in cardiomyocytes. ( A ) Quantitative analysis of all mitotic cardiomyocyte within 3 independent experiments upon control and Pcnt isoform depletion as indicated. For the experiments ≥ 18.000 cardiomyocytes were analyzed per experimental condition. An object classifier was utilized that identifies mitotic cells based on DNA staining patterns. ( B ) Classification of the observed mitotic cardiomyocytes into pro(-meta)phase, metaphase, and ana-/telophase. ( C , E ) Quantification of binucleation ( C ) and mononucleated cardiomyocyte density ( E ). For the experiments ≥ 16023 cardiomyocytes were analyzed per experimental condition. Data are mean ± SD, n = 3, n.s.: p > 0.05, *: p

    Techniques Used: Staining

    Representative examples of different cell cycle stages of cardiomyocytes. ( A ) Representative examples of cardiomyocytes (troponin I) in pro(-meta)phase, metaphase and ana-/telophase. Chromosomes were visualized with DAPI (DNA). Scale bars: 20 μm. ( B ) Representative examples of mono- and binucleated cardiomyocytes (troponin I). Nuclei were visualized with DAPI (DNA). White arrows: cardiomyocyte nuclei. Scale bars: 50 μm.
    Figure Legend Snippet: Representative examples of different cell cycle stages of cardiomyocytes. ( A ) Representative examples of cardiomyocytes (troponin I) in pro(-meta)phase, metaphase and ana-/telophase. Chromosomes were visualized with DAPI (DNA). Scale bars: 20 μm. ( B ) Representative examples of mono- and binucleated cardiomyocytes (troponin I). Nuclei were visualized with DAPI (DNA). White arrows: cardiomyocyte nuclei. Scale bars: 50 μm.

    Techniques Used:

    Effect of Pcnt depletion on centriole configuration. ( A ) Representative examples of P3 cardiomyocytes (troponin I) with paired and split centrioles detected by immunofluorescence analysis of Pcnt S and Pcnt B (Pcnt B+S). Nuclei were visualized with DAPI (DNA). ( B ) Quantitative analysis of cardiomyocytes with paired centrioles for control cells and upon Pcnt isoform depletion as indicated. ( C ) Bayesian posterior distribution for the results in b. Scale bars: 10 μm. Yellow arrows: centrioles/centrosomes. Data are mean ± SD, n = 3, n.s.: p > 0.05, **: p
    Figure Legend Snippet: Effect of Pcnt depletion on centriole configuration. ( A ) Representative examples of P3 cardiomyocytes (troponin I) with paired and split centrioles detected by immunofluorescence analysis of Pcnt S and Pcnt B (Pcnt B+S). Nuclei were visualized with DAPI (DNA). ( B ) Quantitative analysis of cardiomyocytes with paired centrioles for control cells and upon Pcnt isoform depletion as indicated. ( C ) Bayesian posterior distribution for the results in b. Scale bars: 10 μm. Yellow arrows: centrioles/centrosomes. Data are mean ± SD, n = 3, n.s.: p > 0.05, **: p

    Techniques Used: Immunofluorescence

    Immunofluorescence analysis of Pcnt S and Pcnt B localization. P3 cardiomyocytes were transfected with siRNAs to deplete Pcnt S or Pcnt S + B as indicated. ( A ) Immunofluorescence analysis of Pcnt expression in cardiomyocytes (troponin I) utilizing an antibody binding to both Pcnt S and Pcnt B (Pcnt B+S). Nuclei were visualized with DAPI (DNA). ( B ) Semi-quantitative analysis of median intensity of nuclear Pcnt B+S signal in a. ( C ) Immunofluorescence analysis of Pcnt expression in cardiomyocytes (troponin I) utilizing an antibody binding to both Pcnt S and Pcnt B (Pcnt B+S) and an antibody detecting specifically Pcnt B. Nuclei were visualized with DAPI (DNA). ( D ) Semi-quantitative analysis of maximum intensity of the Pcnt B signal per cardiomyocyte in C as an approximation of the centrosomal Pcnt B signal. Yellow asterisk: cardiomyocyte nucleus. White arrows: nuclear envelope of cardiomyocytes. For the experiments ≥ 2000 cardiomyocytes were analyzed per experimental condition. Scale bars: 50 μm. Data are mean ± SD, n = 3, p
    Figure Legend Snippet: Immunofluorescence analysis of Pcnt S and Pcnt B localization. P3 cardiomyocytes were transfected with siRNAs to deplete Pcnt S or Pcnt S + B as indicated. ( A ) Immunofluorescence analysis of Pcnt expression in cardiomyocytes (troponin I) utilizing an antibody binding to both Pcnt S and Pcnt B (Pcnt B+S). Nuclei were visualized with DAPI (DNA). ( B ) Semi-quantitative analysis of median intensity of nuclear Pcnt B+S signal in a. ( C ) Immunofluorescence analysis of Pcnt expression in cardiomyocytes (troponin I) utilizing an antibody binding to both Pcnt S and Pcnt B (Pcnt B+S) and an antibody detecting specifically Pcnt B. Nuclei were visualized with DAPI (DNA). ( D ) Semi-quantitative analysis of maximum intensity of the Pcnt B signal per cardiomyocyte in C as an approximation of the centrosomal Pcnt B signal. Yellow asterisk: cardiomyocyte nucleus. White arrows: nuclear envelope of cardiomyocytes. For the experiments ≥ 2000 cardiomyocytes were analyzed per experimental condition. Scale bars: 50 μm. Data are mean ± SD, n = 3, p

    Techniques Used: Immunofluorescence, Transfection, Expressing, Binding Assay

    Depletion of Pcnt S increases cell-cycle activity and DNA synthesis in postnatal cardiomyocytes. ( A , D ) Representative examples of P3 cardiomyocytes (troponin I) stained for Ki67 ( A ) and incorporated EdU ( D ). Nuclei were visualized with DAPI (DNA). ( B , E ) Quantitative analysis of Ki67 + ( B ) and EdU + ( E ) cardiomyocytes upon control and Pcnt isoform depletion as indicated. ( C , F ) Bayesian posterior distribution for the data in b and e. Scale bars: 50 μm. White arrows: cardiomyocyte nuclei. Data are mean ± SD, **: p
    Figure Legend Snippet: Depletion of Pcnt S increases cell-cycle activity and DNA synthesis in postnatal cardiomyocytes. ( A , D ) Representative examples of P3 cardiomyocytes (troponin I) stained for Ki67 ( A ) and incorporated EdU ( D ). Nuclei were visualized with DAPI (DNA). ( B , E ) Quantitative analysis of Ki67 + ( B ) and EdU + ( E ) cardiomyocytes upon control and Pcnt isoform depletion as indicated. ( C , F ) Bayesian posterior distribution for the data in b and e. Scale bars: 50 μm. White arrows: cardiomyocyte nuclei. Data are mean ± SD, **: p

    Techniques Used: Activity Assay, DNA Synthesis, Staining

    5) Product Images from "Experimental data of labeling the heart and cardiac cultures with a retrograde tracer in vitro and in vivo"

    Article Title: Experimental data of labeling the heart and cardiac cultures with a retrograde tracer in vitro and in vivo

    Journal: Data in Brief

    doi: 10.1016/j.dib.2021.106834

    Imaging data of Di-8-ANEPPQ labeled primary neuronal and cardiac cells in vitro The Di-8-ANEPPQ dye labeling efficiency was determined by flourescent imaging of the primary NG neurons (A) or primary neonatal cardiomyocytes (B) imaged by floresence microscopy. Di-8-ANEPPQ: green, scale bar: 100 µm.
    Figure Legend Snippet: Imaging data of Di-8-ANEPPQ labeled primary neuronal and cardiac cells in vitro The Di-8-ANEPPQ dye labeling efficiency was determined by flourescent imaging of the primary NG neurons (A) or primary neonatal cardiomyocytes (B) imaged by floresence microscopy. Di-8-ANEPPQ: green, scale bar: 100 µm.

    Techniques Used: Imaging, Labeling, In Vitro, Microscopy

    6) Product Images from "AKAP6 orchestrates the nuclear envelope microtubule-organizing center by linking golgi and nucleus via AKAP9"

    Article Title: AKAP6 orchestrates the nuclear envelope microtubule-organizing center by linking golgi and nucleus via AKAP9

    Journal: eLife

    doi: 10.7554/eLife.61669

    AKAP6 anchors centrosomal proteins to nesprin-1α through its SR domains. ( A ) Schematic representation of AKAP6, Pcnt, and AKAP9. ( B ) Immunostaining of Pcnt (red) or AKAP9 (red), cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in siControl and siAKAP6-treated rat P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 suggesting that the SR domains of AKAP6 are sufficient to bind to the nuclear envelope and to anchor centrosomal proteins. Transfected cells are indicated with a yellow arrowhead. ( C ) Immunoprecipitation to demonstrate the interaction of SR1-3 of AKAP6 with the PACT domain of Pcnt or AKAP9. Lysates from HEK293 cells transfected with GFP-AKAP6-SR1-3 in the absence or presence of FLAG-Pcnt-PACT or FLAG-AKAP9-PACT were immunoprecipitated with an anti-FLAG antibody and analyzed by western blotting with antibodies against GFP and FLAG, as indicated. The experiment was performed three times (n = 3); shown is a representative image. ( D ) Schematic representation of the results in E. +: interaction; -: no interaction. ( E ) Lysates from HEK293 cells co-transfected with FLAG-Pcnt-PACT and the indicated GFP-AKAP6-SR domains were immunoprecipitated with an anti-GFP antibody and analyzed by western blotting with antibodies against FLAG and GFP; as indicated. The experiment was performed three times (n = 3); shown is a representative image. ( F ) Yeast-two-hybrid assay. Interactions were tested by monitoring the growth of yeast cells expressing AKAP6-SR1 fused to the DNA binding domain of GAL4 (BD) and Pcnt-PACT or AKAP9-PACT fused to the GAL4 activation domain (AD) proteins on DBO agar plates (double dropout; SD /-Leu /- Trp) (upper), or on QDO plates (quadruple dropout; SD /-Ade /- His/-Leu/-Trp) (lower). Growth on QDO plates indicate interaction. The experiment was performed twice (n = 2); shown is a representative image. ( G–I ) Immunostaining of ( H ) Pcnt (red) or ( I ) AKAP9, cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1 and subsequent quantification of Pcnt- or AKAP9-positive cardiomyocyte nuclei in transfected cells ( G ) Transfected cells are labeled with a yellow arrowhead. Data are represented as individual biological replicates, together with mean ± SD. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. ****: p
    Figure Legend Snippet: AKAP6 anchors centrosomal proteins to nesprin-1α through its SR domains. ( A ) Schematic representation of AKAP6, Pcnt, and AKAP9. ( B ) Immunostaining of Pcnt (red) or AKAP9 (red), cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in siControl and siAKAP6-treated rat P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 suggesting that the SR domains of AKAP6 are sufficient to bind to the nuclear envelope and to anchor centrosomal proteins. Transfected cells are indicated with a yellow arrowhead. ( C ) Immunoprecipitation to demonstrate the interaction of SR1-3 of AKAP6 with the PACT domain of Pcnt or AKAP9. Lysates from HEK293 cells transfected with GFP-AKAP6-SR1-3 in the absence or presence of FLAG-Pcnt-PACT or FLAG-AKAP9-PACT were immunoprecipitated with an anti-FLAG antibody and analyzed by western blotting with antibodies against GFP and FLAG, as indicated. The experiment was performed three times (n = 3); shown is a representative image. ( D ) Schematic representation of the results in E. +: interaction; -: no interaction. ( E ) Lysates from HEK293 cells co-transfected with FLAG-Pcnt-PACT and the indicated GFP-AKAP6-SR domains were immunoprecipitated with an anti-GFP antibody and analyzed by western blotting with antibodies against FLAG and GFP; as indicated. The experiment was performed three times (n = 3); shown is a representative image. ( F ) Yeast-two-hybrid assay. Interactions were tested by monitoring the growth of yeast cells expressing AKAP6-SR1 fused to the DNA binding domain of GAL4 (BD) and Pcnt-PACT or AKAP9-PACT fused to the GAL4 activation domain (AD) proteins on DBO agar plates (double dropout; SD /-Leu /- Trp) (upper), or on QDO plates (quadruple dropout; SD /-Ade /- His/-Leu/-Trp) (lower). Growth on QDO plates indicate interaction. The experiment was performed twice (n = 2); shown is a representative image. ( G–I ) Immunostaining of ( H ) Pcnt (red) or ( I ) AKAP9, cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1 and subsequent quantification of Pcnt- or AKAP9-positive cardiomyocyte nuclei in transfected cells ( G ) Transfected cells are labeled with a yellow arrowhead. Data are represented as individual biological replicates, together with mean ± SD. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. ****: p

    Techniques Used: Immunostaining, Transfection, Immunoprecipitation, Western Blot, Y2H Assay, Expressing, Binding Assay, Activation Assay, Labeling

    AKAP6 and AKAP9 dependent Golgi localization to the nuclear envelope is required for cell-specific functions in cardiomyocytes and osteoclasts. ( A ) Immunostaining of ANF (green), troponin I (red), and DNA (DAPI) of P3 cardiomyocytes transfected with siControl, siAKAP6, or siAKAP9 and stimulated with vehicle or 100 nM ET-1 for 24 hr. Cells were analyzed for a hypertrophic response (perinuclear ANF expression). ( B ) Quantitative analysis of the percentage of ANF-positive troponin I-positive cardiomyocytes. Data are represented as individual biological replicates, together with mean ± SD from three independent experiments. Statistical analysis was performed with two-way ANOVA with post-hoc Bonferroni comparison. *: p
    Figure Legend Snippet: AKAP6 and AKAP9 dependent Golgi localization to the nuclear envelope is required for cell-specific functions in cardiomyocytes and osteoclasts. ( A ) Immunostaining of ANF (green), troponin I (red), and DNA (DAPI) of P3 cardiomyocytes transfected with siControl, siAKAP6, or siAKAP9 and stimulated with vehicle or 100 nM ET-1 for 24 hr. Cells were analyzed for a hypertrophic response (perinuclear ANF expression). ( B ) Quantitative analysis of the percentage of ANF-positive troponin I-positive cardiomyocytes. Data are represented as individual biological replicates, together with mean ± SD from three independent experiments. Statistical analysis was performed with two-way ANOVA with post-hoc Bonferroni comparison. *: p

    Techniques Used: Immunostaining, Transfection, Expressing

    AKAP6 regulates two pools of microtubules at the nuclear envelope. ( A ) Immunostaining of α-tubulin (green), GM130 (red), AKAP9 (magenta), and DNA (DAPI) in control, as well as in the indicated siRNA-transfected cells, after 2 min of recovery from nocodazole-induced microtubule depolymerization. Asterisks indicate centrosomal MTOC and arrowheads indicate nuclear envelope MTOC. ( B ) Quantification of A as α-tubulin intensity in concentric bands around the nucleus normalized to the total intensity of α-tubulin in the cell. 37 siControl cells, 53 siAKAP9 cells, 34 siPcnt cells and 43 siAKAP9+siPcnt cells were quantified per condition, from two independent experiments. Error bars represent the SD. ( C ) Immunostaining of γ-tubulin (magenta), α-tubulin (green), troponin I (red), and DNA (DAPI) in siRNA-treated cardiomyocytes after 2 min of recovery from nocodazole-induced microtubule depolymerization. ( D ) Quantification of C, as γ-tubulin intensity at the nuclear envelope normalized to siControl-treated cells. Statistical test: one-way ANOVA with post-hoc Bonferroni’s comparison. ****: p
    Figure Legend Snippet: AKAP6 regulates two pools of microtubules at the nuclear envelope. ( A ) Immunostaining of α-tubulin (green), GM130 (red), AKAP9 (magenta), and DNA (DAPI) in control, as well as in the indicated siRNA-transfected cells, after 2 min of recovery from nocodazole-induced microtubule depolymerization. Asterisks indicate centrosomal MTOC and arrowheads indicate nuclear envelope MTOC. ( B ) Quantification of A as α-tubulin intensity in concentric bands around the nucleus normalized to the total intensity of α-tubulin in the cell. 37 siControl cells, 53 siAKAP9 cells, 34 siPcnt cells and 43 siAKAP9+siPcnt cells were quantified per condition, from two independent experiments. Error bars represent the SD. ( C ) Immunostaining of γ-tubulin (magenta), α-tubulin (green), troponin I (red), and DNA (DAPI) in siRNA-treated cardiomyocytes after 2 min of recovery from nocodazole-induced microtubule depolymerization. ( D ) Quantification of C, as γ-tubulin intensity at the nuclear envelope normalized to siControl-treated cells. Statistical test: one-way ANOVA with post-hoc Bonferroni’s comparison. ****: p

    Techniques Used: Immunostaining, Transfection

    AKAP6 anchors the Golgi through AKAP9 to the nuclear envelope. ( A ) Immunostaining of AKAP9 (green), GM130 (red), and DNA (DAPI) in siControl- and siAKAP6-treated cardiomyocytes. ( B ) Quantification of A as the GM130 mean intensity in concentric bands of 0.2 µm around the nuclear edge normalized to the total GM130 mean intensity of the cell. 24 cells were analyzed per condition from three independent experiments. Error bars represent the SD. ( C ) Immunostaining of GM130 (green) and DNA (DAPI) in control, AKAP9- or Pcnt-depleted cardiomyocytes. ( D ) Quantification of C as in B. 24 (siControl), 17 (siAKAP9) and 26 (siPcnt) cells were quantified per condition and pooled from three independent experiments. Error bars represent the SD. ( E ) Immunostaining of GM130 (red), cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1, GFP-AKAP9-PACT or GFP-AKAP6-SR1-3 as control. Transfected cells are labeled with a yellow arrowhead. ( F ) Immunostaining of GM130 (red), AKAP9 (magenta), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 or GFP-AKAP9-AK1b. ( G ) Model representing the tethering of the Golgi to the nuclear envelope through AKAP6 and AKAP9 interaction. AKAP6 bridges AKAP9 to nesprin-1α through its SR domains, while AKAP9 bridges AKAP6 to GM130 through its PACT and N-terminal (AK1b) domains. Scale bars: 10 µm. Underlying data for graphs in panels B and D.
    Figure Legend Snippet: AKAP6 anchors the Golgi through AKAP9 to the nuclear envelope. ( A ) Immunostaining of AKAP9 (green), GM130 (red), and DNA (DAPI) in siControl- and siAKAP6-treated cardiomyocytes. ( B ) Quantification of A as the GM130 mean intensity in concentric bands of 0.2 µm around the nuclear edge normalized to the total GM130 mean intensity of the cell. 24 cells were analyzed per condition from three independent experiments. Error bars represent the SD. ( C ) Immunostaining of GM130 (green) and DNA (DAPI) in control, AKAP9- or Pcnt-depleted cardiomyocytes. ( D ) Quantification of C as in B. 24 (siControl), 17 (siAKAP9) and 26 (siPcnt) cells were quantified per condition and pooled from three independent experiments. Error bars represent the SD. ( E ) Immunostaining of GM130 (red), cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1, GFP-AKAP9-PACT or GFP-AKAP6-SR1-3 as control. Transfected cells are labeled with a yellow arrowhead. ( F ) Immunostaining of GM130 (red), AKAP9 (magenta), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 or GFP-AKAP9-AK1b. ( G ) Model representing the tethering of the Golgi to the nuclear envelope through AKAP6 and AKAP9 interaction. AKAP6 bridges AKAP9 to nesprin-1α through its SR domains, while AKAP9 bridges AKAP6 to GM130 through its PACT and N-terminal (AK1b) domains. Scale bars: 10 µm. Underlying data for graphs in panels B and D.

    Techniques Used: Immunostaining, Transfection, Labeling

    Enhanced centrosomal MTOC activity in AKAP6 and AKAP9 depleted cells. ( A ) Immunostaining of α-tubulin (red) and DAPI in P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 or GFP-AKAP6-SR1 after 2 min of recovery from nocodazole-induced microtubule depolymerization. ( B ) Immunostaining of α-tubulin (green), Pcnt (red), AKAP9 (magenta), and DNA (DAPI) in the indicated siRNA-treated cardiomyocytes after 2 min of recovery from nocodazole-induced microtubule depolymerization; insets: 5 µm. Asterisk indicates the centrosome and arrowheads indicate nuclear envelop MTOC. Scale bars: 10 µm.
    Figure Legend Snippet: Enhanced centrosomal MTOC activity in AKAP6 and AKAP9 depleted cells. ( A ) Immunostaining of α-tubulin (red) and DAPI in P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 or GFP-AKAP6-SR1 after 2 min of recovery from nocodazole-induced microtubule depolymerization. ( B ) Immunostaining of α-tubulin (green), Pcnt (red), AKAP9 (magenta), and DNA (DAPI) in the indicated siRNA-treated cardiomyocytes after 2 min of recovery from nocodazole-induced microtubule depolymerization; insets: 5 µm. Asterisk indicates the centrosome and arrowheads indicate nuclear envelop MTOC. Scale bars: 10 µm.

    Techniques Used: Activity Assay, Immunostaining, Transfection

    AKAP6 expression is associated with PCM1 localization at the nuclear envelope. ( A ) RT-PCR of Akap6 and gapdh utilizing RNA from rat heart samples at different developmental stages as indicated. ( B ) Quantification of A, as Akap6 band intensity normalized to Gapdh band intensity. ( C ) Microarray-based temporal expression profile of Akap6 during rat heart development. ( D ) Immunostaining of AKAP6 (green), PCM1 (red), troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in E15 and P3 rat cardiomyocytes. Scale bars: 10 µm. Underlying data for graphs in panel 1C.
    Figure Legend Snippet: AKAP6 expression is associated with PCM1 localization at the nuclear envelope. ( A ) RT-PCR of Akap6 and gapdh utilizing RNA from rat heart samples at different developmental stages as indicated. ( B ) Quantification of A, as Akap6 band intensity normalized to Gapdh band intensity. ( C ) Microarray-based temporal expression profile of Akap6 during rat heart development. ( D ) Immunostaining of AKAP6 (green), PCM1 (red), troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in E15 and P3 rat cardiomyocytes. Scale bars: 10 µm. Underlying data for graphs in panel 1C.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Microarray, Immunostaining

    AKAP6 is required for centrosomal protein recruitment and MTOC function at the nuclear envelope. ( A–B ) Western blot analysis of ( A ) AKAP6 or ( B ) PCM1 expression levels upon siRNA-mediated depletion of AKAP6. Loading control: α-tubulin. ( C ) Immunostaining of nesprin-1α (green), AKAP6 (red), and DNA (DAPI) in siControl- or si-AKAP6-depleted P3 cardiomyocytes. ( D ) Quantification of C as nuclear intensity normalized to the mean intensity in siControl. Error bars represent SD. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. **** p
    Figure Legend Snippet: AKAP6 is required for centrosomal protein recruitment and MTOC function at the nuclear envelope. ( A–B ) Western blot analysis of ( A ) AKAP6 or ( B ) PCM1 expression levels upon siRNA-mediated depletion of AKAP6. Loading control: α-tubulin. ( C ) Immunostaining of nesprin-1α (green), AKAP6 (red), and DNA (DAPI) in siControl- or si-AKAP6-depleted P3 cardiomyocytes. ( D ) Quantification of C as nuclear intensity normalized to the mean intensity in siControl. Error bars represent SD. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. **** p

    Techniques Used: Western Blot, Expressing, Immunostaining

    7) Product Images from "AKAP6 orchestrates the nuclear envelope microtubule-organizing center by linking golgi and nucleus via AKAP9"

    Article Title: AKAP6 orchestrates the nuclear envelope microtubule-organizing center by linking golgi and nucleus via AKAP9

    Journal: eLife

    doi: 10.7554/eLife.61669

    AKAP6 anchors centrosomal proteins to nesprin-1α through its SR domains. ( A ) Schematic representation of AKAP6, Pcnt, and AKAP9. ( B ) Immunostaining of Pcnt (red) or AKAP9 (red), cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in siControl and siAKAP6-treated rat P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 suggesting that the SR domains of AKAP6 are sufficient to bind to the nuclear envelope and to anchor centrosomal proteins. Transfected cells are indicated with a yellow arrowhead. ( C ) Immunoprecipitation to demonstrate the interaction of SR1-3 of AKAP6 with the PACT domain of Pcnt or AKAP9. Lysates from HEK293 cells transfected with GFP-AKAP6-SR1-3 in the absence or presence of FLAG-Pcnt-PACT or FLAG-AKAP9-PACT were immunoprecipitated with an anti-FLAG antibody and analyzed by western blotting with antibodies against GFP and FLAG, as indicated. The experiment was performed three times (n = 3); shown is a representative image. ( D ) Schematic representation of the results in E. +: interaction; -: no interaction. ( E ) Lysates from HEK293 cells co-transfected with FLAG-Pcnt-PACT and the indicated GFP-AKAP6-SR domains were immunoprecipitated with an anti-GFP antibody and analyzed by western blotting with antibodies against FLAG and GFP; as indicated. The experiment was performed three times (n = 3); shown is a representative image. ( F ) Yeast-two-hybrid assay. Interactions were tested by monitoring the growth of yeast cells expressing AKAP6-SR1 fused to the DNA binding domain of GAL4 (BD) and Pcnt-PACT or AKAP9-PACT fused to the GAL4 activation domain (AD) proteins on DBO agar plates (double dropout; SD /-Leu /- Trp) (upper), or on QDO plates (quadruple dropout; SD /-Ade /- His/-Leu/-Trp) (lower). Growth on QDO plates indicate interaction. The experiment was performed twice (n = 2); shown is a representative image. ( G–I ) Immunostaining of ( H ) Pcnt (red) or ( I ) AKAP9, cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1 and subsequent quantification of Pcnt- or AKAP9-positive cardiomyocyte nuclei in transfected cells ( G ) Transfected cells are labeled with a yellow arrowhead. Data are represented as individual biological replicates, together with mean ± SD. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. ****: p
    Figure Legend Snippet: AKAP6 anchors centrosomal proteins to nesprin-1α through its SR domains. ( A ) Schematic representation of AKAP6, Pcnt, and AKAP9. ( B ) Immunostaining of Pcnt (red) or AKAP9 (red), cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in siControl and siAKAP6-treated rat P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 suggesting that the SR domains of AKAP6 are sufficient to bind to the nuclear envelope and to anchor centrosomal proteins. Transfected cells are indicated with a yellow arrowhead. ( C ) Immunoprecipitation to demonstrate the interaction of SR1-3 of AKAP6 with the PACT domain of Pcnt or AKAP9. Lysates from HEK293 cells transfected with GFP-AKAP6-SR1-3 in the absence or presence of FLAG-Pcnt-PACT or FLAG-AKAP9-PACT were immunoprecipitated with an anti-FLAG antibody and analyzed by western blotting with antibodies against GFP and FLAG, as indicated. The experiment was performed three times (n = 3); shown is a representative image. ( D ) Schematic representation of the results in E. +: interaction; -: no interaction. ( E ) Lysates from HEK293 cells co-transfected with FLAG-Pcnt-PACT and the indicated GFP-AKAP6-SR domains were immunoprecipitated with an anti-GFP antibody and analyzed by western blotting with antibodies against FLAG and GFP; as indicated. The experiment was performed three times (n = 3); shown is a representative image. ( F ) Yeast-two-hybrid assay. Interactions were tested by monitoring the growth of yeast cells expressing AKAP6-SR1 fused to the DNA binding domain of GAL4 (BD) and Pcnt-PACT or AKAP9-PACT fused to the GAL4 activation domain (AD) proteins on DBO agar plates (double dropout; SD /-Leu /- Trp) (upper), or on QDO plates (quadruple dropout; SD /-Ade /- His/-Leu/-Trp) (lower). Growth on QDO plates indicate interaction. The experiment was performed twice (n = 2); shown is a representative image. ( G–I ) Immunostaining of ( H ) Pcnt (red) or ( I ) AKAP9, cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1 and subsequent quantification of Pcnt- or AKAP9-positive cardiomyocyte nuclei in transfected cells ( G ) Transfected cells are labeled with a yellow arrowhead. Data are represented as individual biological replicates, together with mean ± SD. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. ****: p

    Techniques Used: Immunostaining, Transfection, Immunoprecipitation, Western Blot, Y2H Assay, Expressing, Binding Assay, Activation Assay, Labeling

    AKAP6 and AKAP9 dependent Golgi localization to the nuclear envelope is required for cell-specific functions in cardiomyocytes and osteoclasts. ( A ) Immunostaining of ANF (green), troponin I (red), and DNA (DAPI) of P3 cardiomyocytes transfected with siControl, siAKAP6, or siAKAP9 and stimulated with vehicle or 100 nM ET-1 for 24 hr. Cells were analyzed for a hypertrophic response (perinuclear ANF expression). ( B ) Quantitative analysis of the percentage of ANF-positive troponin I-positive cardiomyocytes. Data are represented as individual biological replicates, together with mean ± SD from three independent experiments. Statistical analysis was performed with two-way ANOVA with post-hoc Bonferroni comparison. *: p
    Figure Legend Snippet: AKAP6 and AKAP9 dependent Golgi localization to the nuclear envelope is required for cell-specific functions in cardiomyocytes and osteoclasts. ( A ) Immunostaining of ANF (green), troponin I (red), and DNA (DAPI) of P3 cardiomyocytes transfected with siControl, siAKAP6, or siAKAP9 and stimulated with vehicle or 100 nM ET-1 for 24 hr. Cells were analyzed for a hypertrophic response (perinuclear ANF expression). ( B ) Quantitative analysis of the percentage of ANF-positive troponin I-positive cardiomyocytes. Data are represented as individual biological replicates, together with mean ± SD from three independent experiments. Statistical analysis was performed with two-way ANOVA with post-hoc Bonferroni comparison. *: p

    Techniques Used: Immunostaining, Transfection, Expressing

    AKAP6 regulates two pools of microtubules at the nuclear envelope. ( A ) Immunostaining of α-tubulin (green), GM130 (red), AKAP9 (magenta), and DNA (DAPI) in control, as well as in the indicated siRNA-transfected cells, after 2 min of recovery from nocodazole-induced microtubule depolymerization. Asterisks indicate centrosomal MTOC and arrowheads indicate nuclear envelope MTOC. ( B ) Quantification of A as α-tubulin intensity in concentric bands around the nucleus normalized to the total intensity of α-tubulin in the cell. 37 siControl cells, 53 siAKAP9 cells, 34 siPcnt cells and 43 siAKAP9+siPcnt cells were quantified per condition, from two independent experiments. Error bars represent the SD. ( C ) Immunostaining of γ-tubulin (magenta), α-tubulin (green), troponin I (red), and DNA (DAPI) in siRNA-treated cardiomyocytes after 2 min of recovery from nocodazole-induced microtubule depolymerization. ( D ) Quantification of C, as γ-tubulin intensity at the nuclear envelope normalized to siControl-treated cells. Statistical test: one-way ANOVA with post-hoc Bonferroni’s comparison. ****: p
    Figure Legend Snippet: AKAP6 regulates two pools of microtubules at the nuclear envelope. ( A ) Immunostaining of α-tubulin (green), GM130 (red), AKAP9 (magenta), and DNA (DAPI) in control, as well as in the indicated siRNA-transfected cells, after 2 min of recovery from nocodazole-induced microtubule depolymerization. Asterisks indicate centrosomal MTOC and arrowheads indicate nuclear envelope MTOC. ( B ) Quantification of A as α-tubulin intensity in concentric bands around the nucleus normalized to the total intensity of α-tubulin in the cell. 37 siControl cells, 53 siAKAP9 cells, 34 siPcnt cells and 43 siAKAP9+siPcnt cells were quantified per condition, from two independent experiments. Error bars represent the SD. ( C ) Immunostaining of γ-tubulin (magenta), α-tubulin (green), troponin I (red), and DNA (DAPI) in siRNA-treated cardiomyocytes after 2 min of recovery from nocodazole-induced microtubule depolymerization. ( D ) Quantification of C, as γ-tubulin intensity at the nuclear envelope normalized to siControl-treated cells. Statistical test: one-way ANOVA with post-hoc Bonferroni’s comparison. ****: p

    Techniques Used: Immunostaining, Transfection

    AKAP6 anchors the Golgi through AKAP9 to the nuclear envelope. ( A ) Immunostaining of AKAP9 (green), GM130 (red), and DNA (DAPI) in siControl- and siAKAP6-treated cardiomyocytes. ( B ) Quantification of A as the GM130 mean intensity in concentric bands of 0.2 µm around the nuclear edge normalized to the total GM130 mean intensity of the cell. 24 cells were analyzed per condition from three independent experiments. Error bars represent the SD. ( C ) Immunostaining of GM130 (green) and DNA (DAPI) in control, AKAP9- or Pcnt-depleted cardiomyocytes. ( D ) Quantification of C as in B. 24 (siControl), 17 (siAKAP9) and 26 (siPcnt) cells were quantified per condition and pooled from three independent experiments. Error bars represent the SD. ( E ) Immunostaining of GM130 (red), cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1, GFP-AKAP9-PACT or GFP-AKAP6-SR1-3 as control. Transfected cells are labeled with a yellow arrowhead. ( F ) Immunostaining of GM130 (red), AKAP9 (magenta), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 or GFP-AKAP9-AK1b. ( G ) Model representing the tethering of the Golgi to the nuclear envelope through AKAP6 and AKAP9 interaction. AKAP6 bridges AKAP9 to nesprin-1α through its SR domains, while AKAP9 bridges AKAP6 to GM130 through its PACT and N-terminal (AK1b) domains. Scale bars: 10 µm. Underlying data for graphs in panels B and D.
    Figure Legend Snippet: AKAP6 anchors the Golgi through AKAP9 to the nuclear envelope. ( A ) Immunostaining of AKAP9 (green), GM130 (red), and DNA (DAPI) in siControl- and siAKAP6-treated cardiomyocytes. ( B ) Quantification of A as the GM130 mean intensity in concentric bands of 0.2 µm around the nuclear edge normalized to the total GM130 mean intensity of the cell. 24 cells were analyzed per condition from three independent experiments. Error bars represent the SD. ( C ) Immunostaining of GM130 (green) and DNA (DAPI) in control, AKAP9- or Pcnt-depleted cardiomyocytes. ( D ) Quantification of C as in B. 24 (siControl), 17 (siAKAP9) and 26 (siPcnt) cells were quantified per condition and pooled from three independent experiments. Error bars represent the SD. ( E ) Immunostaining of GM130 (red), cardiac troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1, GFP-AKAP9-PACT or GFP-AKAP6-SR1-3 as control. Transfected cells are labeled with a yellow arrowhead. ( F ) Immunostaining of GM130 (red), AKAP9 (magenta), and DNA (DAPI) in P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 or GFP-AKAP9-AK1b. ( G ) Model representing the tethering of the Golgi to the nuclear envelope through AKAP6 and AKAP9 interaction. AKAP6 bridges AKAP9 to nesprin-1α through its SR domains, while AKAP9 bridges AKAP6 to GM130 through its PACT and N-terminal (AK1b) domains. Scale bars: 10 µm. Underlying data for graphs in panels B and D.

    Techniques Used: Immunostaining, Transfection, Labeling

    Enhanced centrosomal MTOC activity in AKAP6 and AKAP9 depleted cells. ( A ) Immunostaining of α-tubulin (red) and DAPI in P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 or GFP-AKAP6-SR1 after 2 min of recovery from nocodazole-induced microtubule depolymerization. ( B ) Immunostaining of α-tubulin (green), Pcnt (red), AKAP9 (magenta), and DNA (DAPI) in the indicated siRNA-treated cardiomyocytes after 2 min of recovery from nocodazole-induced microtubule depolymerization; insets: 5 µm. Asterisk indicates the centrosome and arrowheads indicate nuclear envelop MTOC. Scale bars: 10 µm.
    Figure Legend Snippet: Enhanced centrosomal MTOC activity in AKAP6 and AKAP9 depleted cells. ( A ) Immunostaining of α-tubulin (red) and DAPI in P3 cardiomyocytes transfected with GFP-AKAP6-SR1-3 or GFP-AKAP6-SR1 after 2 min of recovery from nocodazole-induced microtubule depolymerization. ( B ) Immunostaining of α-tubulin (green), Pcnt (red), AKAP9 (magenta), and DNA (DAPI) in the indicated siRNA-treated cardiomyocytes after 2 min of recovery from nocodazole-induced microtubule depolymerization; insets: 5 µm. Asterisk indicates the centrosome and arrowheads indicate nuclear envelop MTOC. Scale bars: 10 µm.

    Techniques Used: Activity Assay, Immunostaining, Transfection

    AKAP6 expression is associated with PCM1 localization at the nuclear envelope. ( A ) RT-PCR of Akap6 and gapdh utilizing RNA from rat heart samples at different developmental stages as indicated. ( B ) Quantification of A, as Akap6 band intensity normalized to Gapdh band intensity. ( C ) Microarray-based temporal expression profile of Akap6 during rat heart development. ( D ) Immunostaining of AKAP6 (green), PCM1 (red), troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in E15 and P3 rat cardiomyocytes. Scale bars: 10 µm. Underlying data for graphs in panel 1C.
    Figure Legend Snippet: AKAP6 expression is associated with PCM1 localization at the nuclear envelope. ( A ) RT-PCR of Akap6 and gapdh utilizing RNA from rat heart samples at different developmental stages as indicated. ( B ) Quantification of A, as Akap6 band intensity normalized to Gapdh band intensity. ( C ) Microarray-based temporal expression profile of Akap6 during rat heart development. ( D ) Immunostaining of AKAP6 (green), PCM1 (red), troponin I (magenta, cardiomyocyte-specific), and DNA (DAPI) in E15 and P3 rat cardiomyocytes. Scale bars: 10 µm. Underlying data for graphs in panel 1C.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Microarray, Immunostaining

    AKAP6 is required for centrosomal protein recruitment and MTOC function at the nuclear envelope. ( A–B ) Western blot analysis of ( A ) AKAP6 or ( B ) PCM1 expression levels upon siRNA-mediated depletion of AKAP6. Loading control: α-tubulin. ( C ) Immunostaining of nesprin-1α (green), AKAP6 (red), and DNA (DAPI) in siControl- or si-AKAP6-depleted P3 cardiomyocytes. ( D ) Quantification of C as nuclear intensity normalized to the mean intensity in siControl. Error bars represent SD. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. **** p
    Figure Legend Snippet: AKAP6 is required for centrosomal protein recruitment and MTOC function at the nuclear envelope. ( A–B ) Western blot analysis of ( A ) AKAP6 or ( B ) PCM1 expression levels upon siRNA-mediated depletion of AKAP6. Loading control: α-tubulin. ( C ) Immunostaining of nesprin-1α (green), AKAP6 (red), and DNA (DAPI) in siControl- or si-AKAP6-depleted P3 cardiomyocytes. ( D ) Quantification of C as nuclear intensity normalized to the mean intensity in siControl. Error bars represent SD. Statistical test: two-way ANOVA with post-hoc Bonferroni comparison. **** p

    Techniques Used: Western Blot, Expressing, Immunostaining

    8) Product Images from "Angiotensin II induces apoptosis in human induced pluripotent stem cell-derived cardiomyocytes"

    Article Title: Angiotensin II induces apoptosis in human induced pluripotent stem cell-derived cardiomyocytes

    Journal: bioRxiv

    doi: 10.1101/2020.03.19.998344

    AT1 was the major angiotensin II receptor in iPSC-CM. RNA-seq data about AGTR1, AGTR2, ACE, REN, AGT and GAPDH genes from a) GSE116547 dataset and b) GSE137920 dataset were re-analyzed. Transcripts per million (TPM) were used to evaluate the expression levels of each gene. D+number denoted the days during cardiomyocyte differentiation.
    Figure Legend Snippet: AT1 was the major angiotensin II receptor in iPSC-CM. RNA-seq data about AGTR1, AGTR2, ACE, REN, AGT and GAPDH genes from a) GSE116547 dataset and b) GSE137920 dataset were re-analyzed. Transcripts per million (TPM) were used to evaluate the expression levels of each gene. D+number denoted the days during cardiomyocyte differentiation.

    Techniques Used: RNA Sequencing Assay, Expressing

    Cell viability was decreased after long-term Ang II treatment. The effect of a series of Ang II concentrations (1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM) to iPSC-CM was evaluated by PrestoBlue reagent. The relative fluorescence unit (RFU) fold changes from untreated cardiomyocytes to Ang II-treated cardiomyocytes were calculated in a), short-term (24 hours and 48 hours) treatment set and b), long-term (6 days and 10 days) treatment set (n=7). #, p
    Figure Legend Snippet: Cell viability was decreased after long-term Ang II treatment. The effect of a series of Ang II concentrations (1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM) to iPSC-CM was evaluated by PrestoBlue reagent. The relative fluorescence unit (RFU) fold changes from untreated cardiomyocytes to Ang II-treated cardiomyocytes were calculated in a), short-term (24 hours and 48 hours) treatment set and b), long-term (6 days and 10 days) treatment set (n=7). #, p

    Techniques Used: Fluorescence

    Long-term Ang II treatment caused apoptosis in iPSC-CM. a) Representative fluorescence-activated cell sorting (FACS) analyses of apoptosis marker Annexin V in iPSC-CM with 0 nM (untreated), 100 μM and 1 mM Ang II treatment for 10 days. In the unstained group, the DPBS buffer was used instead of the Annexin V staining reagent. b) The proportion of Annexin V-positive cells (PAP) represented the apoptosis status of iPSC-CM. The fold changes of PAP from untreated cardiomyocytes to Ang II-treated cardiomyocytes at different concentrations were calculated (n=3). **, p
    Figure Legend Snippet: Long-term Ang II treatment caused apoptosis in iPSC-CM. a) Representative fluorescence-activated cell sorting (FACS) analyses of apoptosis marker Annexin V in iPSC-CM with 0 nM (untreated), 100 μM and 1 mM Ang II treatment for 10 days. In the unstained group, the DPBS buffer was used instead of the Annexin V staining reagent. b) The proportion of Annexin V-positive cells (PAP) represented the apoptosis status of iPSC-CM. The fold changes of PAP from untreated cardiomyocytes to Ang II-treated cardiomyocytes at different concentrations were calculated (n=3). **, p

    Techniques Used: Fluorescence, FACS, Marker, Staining

    9) Product Images from "Naturally Prefabricated Marine Biomaterials: Isolation and Applications of Flat Chitinous 3D Scaffolds from Ianthella labyrinthus (Demospongiae: Verongiida)"

    Article Title: Naturally Prefabricated Marine Biomaterials: Isolation and Applications of Flat Chitinous 3D Scaffolds from Ianthella labyrinthus (Demospongiae: Verongiida)

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms20205105

    Evaluation of I. labyrinthus structures to culture iPSC-derived cardiomyocytes. ( A ) Schematic image of sponge structures cultured in transwell plates with low medium levels, 8-week-old iPSC-CMs were supplied by medium absorbed by the sponge structure and cultured in uncoated and Geltrex ® -coated sponge scaffolds for 20 days. ( B ) Microscopic images of sponge scaffold. iPSC-CMs were visualized using phase contrast microscopy. Images from two experiments performed using iPSC-CMs from different healthy donors. See also respective video files in the Supplementary Materials .
    Figure Legend Snippet: Evaluation of I. labyrinthus structures to culture iPSC-derived cardiomyocytes. ( A ) Schematic image of sponge structures cultured in transwell plates with low medium levels, 8-week-old iPSC-CMs were supplied by medium absorbed by the sponge structure and cultured in uncoated and Geltrex ® -coated sponge scaffolds for 20 days. ( B ) Microscopic images of sponge scaffold. iPSC-CMs were visualized using phase contrast microscopy. Images from two experiments performed using iPSC-CMs from different healthy donors. See also respective video files in the Supplementary Materials .

    Techniques Used: Derivative Assay, Cell Culture, Microscopy

    10) Product Images from "Generation of Functional Cardiomyocytes from the Synoviocytes of Patients with Rheumatoid Arthritis via Induced Pluripotent Stem Cells"

    Article Title: Generation of Functional Cardiomyocytes from the Synoviocytes of Patients with Rheumatoid Arthritis via Induced Pluripotent Stem Cells

    Journal: Scientific Reports

    doi: 10.1038/srep32669

    Differentiation of patient-specific iPSCs into cardiomyocytes. ( a ) Beating cardiomyocytes were generated from control-iPSCs and RA-iPSCs after 12 days of cardiac differentiation. ( b ) The cellular structure and expression of cardiac markers in iPSC-derived cardiomyocytes were confirmed by immunofluorescence staining and confocal microscopy. Control-iPSC-CMs and RA-iPSC-CMs both showed characteristic sarcomeric structures and positive staining with antibodies against SA-actinin, TNNT2 and MLC2a (scale bar, 200 μm). ( c ) The mRNA level of a cardiac-specific gene, TNNT2, before and after cardiac differentiation in various cells was determined by quantitative reverse transcription-polymerase chain reaction. Data represent mean values determined in three independent experiments ± SEM. ** P
    Figure Legend Snippet: Differentiation of patient-specific iPSCs into cardiomyocytes. ( a ) Beating cardiomyocytes were generated from control-iPSCs and RA-iPSCs after 12 days of cardiac differentiation. ( b ) The cellular structure and expression of cardiac markers in iPSC-derived cardiomyocytes were confirmed by immunofluorescence staining and confocal microscopy. Control-iPSC-CMs and RA-iPSC-CMs both showed characteristic sarcomeric structures and positive staining with antibodies against SA-actinin, TNNT2 and MLC2a (scale bar, 200 μm). ( c ) The mRNA level of a cardiac-specific gene, TNNT2, before and after cardiac differentiation in various cells was determined by quantitative reverse transcription-polymerase chain reaction. Data represent mean values determined in three independent experiments ± SEM. ** P

    Techniques Used: Generated, Expressing, Derivative Assay, Immunofluorescence, Staining, Confocal Microscopy, Reverse Transcription Polymerase Chain Reaction

    Characterisation of cardiomyocytes derived from patient-specific iPSCs. ( a ) Representative line-scan images and spontaneous calcium signalling of RA-iPSC-CMs and control-iPSC-CMs showed regular calcium transients. ( b ) The calcium-handling properties of RA-iPSC-CMs, OA-iPSC-CMs and control-iPSC-CMs were compared by examining calcium-handling parameters. ( c ) Contractility measurements were recorded on single, spontaneously beating cardiomyocytes derived from RA-iPSCs and control-iPSCs. ( d ) Expression of calcium signalling-related genes was measured in iPSC-CMs using quantitative reverse transcription-polymerase chain reaction. Data represent mean values determined in three independent experiments ± SEM. NS, not significant; * P
    Figure Legend Snippet: Characterisation of cardiomyocytes derived from patient-specific iPSCs. ( a ) Representative line-scan images and spontaneous calcium signalling of RA-iPSC-CMs and control-iPSC-CMs showed regular calcium transients. ( b ) The calcium-handling properties of RA-iPSC-CMs, OA-iPSC-CMs and control-iPSC-CMs were compared by examining calcium-handling parameters. ( c ) Contractility measurements were recorded on single, spontaneously beating cardiomyocytes derived from RA-iPSCs and control-iPSCs. ( d ) Expression of calcium signalling-related genes was measured in iPSC-CMs using quantitative reverse transcription-polymerase chain reaction. Data represent mean values determined in three independent experiments ± SEM. NS, not significant; * P

    Techniques Used: Derivative Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

    11) Product Images from "Liver Cell Line Derived Conditioned Medium Enhances Myofibril Organization of Primary Rat Cardiomyocytes"

    Article Title: Liver Cell Line Derived Conditioned Medium Enhances Myofibril Organization of Primary Rat Cardiomyocytes

    Journal: Molecules and Cells

    doi: 10.1007/s10059-012-0019-0

    Cardiomyocyte proliferation in different culture conditions and the morphological and immunocytochemical characterization of cardiomyocytes with different initial cell seeding densities: (A) Time course images of cardiomyocytes in normal cardiomyocyte
    Figure Legend Snippet: Cardiomyocyte proliferation in different culture conditions and the morphological and immunocytochemical characterization of cardiomyocytes with different initial cell seeding densities: (A) Time course images of cardiomyocytes in normal cardiomyocyte

    Techniques Used:

    The confocal scanning microscope images of cardiomyocyte beating mediated by calcium fluctuation using calcium indicator (Fluo-4 AM): (A) Time-lapsed images of calcium fluctuation in cardiomyocyte culture with normal cardiomyocyte medium. (B) Time-lapsed
    Figure Legend Snippet: The confocal scanning microscope images of cardiomyocyte beating mediated by calcium fluctuation using calcium indicator (Fluo-4 AM): (A) Time-lapsed images of calcium fluctuation in cardiomyocyte culture with normal cardiomyocyte medium. (B) Time-lapsed

    Techniques Used: Microscopy

    The characterization of the distribution of cardiac fibroblasts and cardiomyocytes, and the proliferation o f cardiac fibroblasts in different culture conditions (A) Immunocytochemical staining of cardiomyocytes and cardiac fibroblasts in various culture
    Figure Legend Snippet: The characterization of the distribution of cardiac fibroblasts and cardiomyocytes, and the proliferation o f cardiac fibroblasts in different culture conditions (A) Immunocytochemical staining of cardiomyocytes and cardiac fibroblasts in various culture

    Techniques Used: Staining

    Morphological and immunocytochemical characterization of primary neonatal rat cardiomyocytes in various culture conditions: (A) FACS analysis for β-MHC positive cardiomyocyte population after purification by Percoll gradient centrifuge. (B) Morphology
    Figure Legend Snippet: Morphological and immunocytochemical characterization of primary neonatal rat cardiomyocytes in various culture conditions: (A) FACS analysis for β-MHC positive cardiomyocyte population after purification by Percoll gradient centrifuge. (B) Morphology

    Techniques Used: FACS, Purification, Gradient Centrifugation

    The effect of soluble factors on cardiomyocyte morphology and cardio-myofibril organization: (A) The effect of reduced FBS concentration on myofibril organization. Cells were cultured with 5% FBS. Scale bars are 100 μm. (B) The effect of LIF,
    Figure Legend Snippet: The effect of soluble factors on cardiomyocyte morphology and cardio-myofibril organization: (A) The effect of reduced FBS concentration on myofibril organization. Cells were cultured with 5% FBS. Scale bars are 100 μm. (B) The effect of LIF,

    Techniques Used: Concentration Assay, Cell Culture

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    Thermo Fisher medium 199
    Medium 199, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cardiomyocyte medium
    Chronic 3HB exposure induces CD36-mediated lipid accumulation in <t>cardiomyocytes.</t> ARCMs were cultured for 24 h in either low palmitate <t>medium</t> (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB), or LP with 3HB in the presence of 4*KLR (3HB/4*KLR). ( A,B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. ( A ) Assessment of cell-surface CD36 using biotinylation assay. For this, CD36 was detected by Western blotting in biotin immunoprecipitations and total cell lysates and subsequently quantified (n = 6). ( B ) [14C]palmitate uptake (n = 6). ( C ) Triacylglycerol contents (n = 7). Bar values are means ± SEM. * p
    Cardiomyocyte Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Chronic 3HB exposure induces CD36-mediated lipid accumulation in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB), or LP with 3HB in the presence of 4*KLR (3HB/4*KLR). ( A,B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. ( A ) Assessment of cell-surface CD36 using biotinylation assay. For this, CD36 was detected by Western blotting in biotin immunoprecipitations and total cell lysates and subsequently quantified (n = 6). ( B ) [14C]palmitate uptake (n = 6). ( C ) Triacylglycerol contents (n = 7). Bar values are means ± SEM. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly—Rescue by Specific Amino Acid Supplementation

    doi: 10.3390/ijms232112909

    Figure Lengend Snippet: Chronic 3HB exposure induces CD36-mediated lipid accumulation in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB), or LP with 3HB in the presence of 4*KLR (3HB/4*KLR). ( A,B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. ( A ) Assessment of cell-surface CD36 using biotinylation assay. For this, CD36 was detected by Western blotting in biotin immunoprecipitations and total cell lysates and subsequently quantified (n = 6). ( B ) [14C]palmitate uptake (n = 6). ( C ) Triacylglycerol contents (n = 7). Bar values are means ± SEM. * p

    Article Snippet: At day 1 of differentiation, Essential 8 medium was removed and replaced with cardiomyocyte medium a (Thermofisher Scientific, Miami, FL, USA).

    Techniques: Cell Culture, Incubation, Cell Surface Biotinylation Assay, Western Blot

    Chronic 3HB exposure prevents insulin-stimulated GLUT4 translocation and glucose uptake in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB); LP with 3HB in the presence of 4*KLR (3HB/4*KLR); or LP with 3 mM acetoacetate (AcAc). ( A , B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. ( A ) Microscopical assay of cell-surface GLUT4. aRCMs were transduced for 24 h with an adenoviral vector containing an HA-GLUT4-GFP fusion protein before the start of the culturing. Non-permeabilized cells were anti-HA immunostained. The nuclei are stained in blue (DAPI). Representative microscopical images are displayed (the scale bar is 50 µm). The ratios of red (HA-tag) and green (GFP) intensity per pixel were quantified by Image J (n = 6). ( B ) Biotinylation assay. Representative Western blot and quantification of insulin-regulated aminopeptidase (IRAP, which reflects GLUT4 translocation) in biotin-immunoprecipitations and in total lysates (n = 6). ( C ) [3H]Deoxyglucose uptake (n ≥ 6). Bar values are means ± SEM. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly—Rescue by Specific Amino Acid Supplementation

    doi: 10.3390/ijms232112909

    Figure Lengend Snippet: Chronic 3HB exposure prevents insulin-stimulated GLUT4 translocation and glucose uptake in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB); LP with 3HB in the presence of 4*KLR (3HB/4*KLR); or LP with 3 mM acetoacetate (AcAc). ( A , B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. ( A ) Microscopical assay of cell-surface GLUT4. aRCMs were transduced for 24 h with an adenoviral vector containing an HA-GLUT4-GFP fusion protein before the start of the culturing. Non-permeabilized cells were anti-HA immunostained. The nuclei are stained in blue (DAPI). Representative microscopical images are displayed (the scale bar is 50 µm). The ratios of red (HA-tag) and green (GFP) intensity per pixel were quantified by Image J (n = 6). ( B ) Biotinylation assay. Representative Western blot and quantification of insulin-regulated aminopeptidase (IRAP, which reflects GLUT4 translocation) in biotin-immunoprecipitations and in total lysates (n = 6). ( C ) [3H]Deoxyglucose uptake (n ≥ 6). Bar values are means ± SEM. * p

    Article Snippet: At day 1 of differentiation, Essential 8 medium was removed and replaced with cardiomyocyte medium a (Thermofisher Scientific, Miami, FL, USA).

    Techniques: Translocation Assay, Cell Culture, Incubation, Plasmid Preparation, Staining, Cell Surface Biotinylation Assay, Western Blot

    Chronic 3HB exposure reduces v-ATPase activity and insulin sensitivity in human cardiomyocytes. HiPSC-CMs were cultured for 24 h in either control medium (Ctrl), high palmitate medium (HP), Ctrl with 3mM Acetoacetate (AcAc), Ctrl with 3mM 3-β-hydroxybutyrate (3HB), 3HB with supplementation of the 4*KLR mixture (3HB/4*KLR), and with 100 nM Baf (3HB/4*KLR/BafA). ( A ) V-ATPase function in hiPSC-CMs: after culturing, cells were subjected to the [3H] CHLQ accumulation assay (n = 9). ( B , C ) After 24 h, cells were short-term (30 min) incubated without/with 200 nM insulin. ( B ) [14C]palmitate uptake (n = 6). ( C ) [3H] deoxyglucose uptake (n = 3). ( D ) Insulin signaling: Western analysis of phosphorylation of Akt and S6 at Ser473 and Ser235/236, respectively. Representative blots of pAKT and pS6 and Caveolin-3 (loading control) are displayed (n = 4). Bar values are means ± SEM. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly—Rescue by Specific Amino Acid Supplementation

    doi: 10.3390/ijms232112909

    Figure Lengend Snippet: Chronic 3HB exposure reduces v-ATPase activity and insulin sensitivity in human cardiomyocytes. HiPSC-CMs were cultured for 24 h in either control medium (Ctrl), high palmitate medium (HP), Ctrl with 3mM Acetoacetate (AcAc), Ctrl with 3mM 3-β-hydroxybutyrate (3HB), 3HB with supplementation of the 4*KLR mixture (3HB/4*KLR), and with 100 nM Baf (3HB/4*KLR/BafA). ( A ) V-ATPase function in hiPSC-CMs: after culturing, cells were subjected to the [3H] CHLQ accumulation assay (n = 9). ( B , C ) After 24 h, cells were short-term (30 min) incubated without/with 200 nM insulin. ( B ) [14C]palmitate uptake (n = 6). ( C ) [3H] deoxyglucose uptake (n = 3). ( D ) Insulin signaling: Western analysis of phosphorylation of Akt and S6 at Ser473 and Ser235/236, respectively. Representative blots of pAKT and pS6 and Caveolin-3 (loading control) are displayed (n = 4). Bar values are means ± SEM. * p

    Article Snippet: At day 1 of differentiation, Essential 8 medium was removed and replaced with cardiomyocyte medium a (Thermofisher Scientific, Miami, FL, USA).

    Techniques: Activity Assay, Cell Culture, Incubation, Western Blot

    Chronic 3HB exposure causes v-ATPase disassembly and de-activation in cardiomyocytes. ( A ) 3HB exposure results in the loss of v-ATPase activity (measured as [3H]chloroquine accumulation) in cardiomyocytes: aRCMs were cultured for 24 h in either low palmitate medium (LP; control condition); LP with 100 nM bafilomycin-A (BafA), LP with 3 mM acetoacetate (AcAc); LP with different concentrations of 3-β-hydroxybutyrate (3HB; 1mM, 3 mM, and 9 mM), high palmitate medium (HP), HP with 3mM AcAc (HP/AcAc), or HP with 3mM 3HB (HP/3HB). Directly after the culturing, cells were used for the [3H]chloroquine accumulation assay. (n = 5). ( B ) Amino acids prevent 3HB-induced v-ATPase inhibition in cardiomyocytes: aRCMs were incubated for 24 h in either LP, HP, BafA, LP supplemented with 1.56 mM Lys, 1.84 mM Leu and 1.36 mM Arg, (LP/4*KLR; for explanation see Section 4.2 ), 3HB (3 mM), 3HB supplemented with the 4*KLR mix (3HB/4*KLR). Directly after the culturing, cells were used for the [3H]chloroquine accumulation assay (n = 5). ( C – F ) 3HB exposure induces v-ATPase disassembly: HL-1 cells were incubated for 24 h in either control (Ctrl) medium, HP medium, or Ctrl medium with 3 mM 3HB (3HB). ( C ) Subcellular fractionation: cytoplasmic fractions (C) and membrane fractions (M) were analyzed by Western blotting of v-ATPase subunits B2 (V1-B2) and d1 (V0-d1), after which the signals were quantified. For V1-B2, the signal ratio of membrane fraction/cytoplasm in control condition is set at 1.0. For V0-d1, the signal density in the membrane fraction is 1.0. The quantified signals in the other conditions are expressed as multiples. Representative blots are displayed. Caveolin-3 (Cav-3) and GAPDH: loading controls for membrane and cytoplasmic fraction, respectively. (n = 4). ( D – F ) Co-Immunoprecipitation (Co-IP) of V1, V0, and mTORC1. ( D ) IP with V1-B2, ( E ) IP with V0-d1. ( F ) IP with mTORC1. Immunoprecipitates were blotted with antibodies against mTORC1, V0-a2, V0-d1, and V1-B2, after which the signals were quantified. Representative Western blots are displayed. (n = 3). Bar values are means ± SEM. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly—Rescue by Specific Amino Acid Supplementation

    doi: 10.3390/ijms232112909

    Figure Lengend Snippet: Chronic 3HB exposure causes v-ATPase disassembly and de-activation in cardiomyocytes. ( A ) 3HB exposure results in the loss of v-ATPase activity (measured as [3H]chloroquine accumulation) in cardiomyocytes: aRCMs were cultured for 24 h in either low palmitate medium (LP; control condition); LP with 100 nM bafilomycin-A (BafA), LP with 3 mM acetoacetate (AcAc); LP with different concentrations of 3-β-hydroxybutyrate (3HB; 1mM, 3 mM, and 9 mM), high palmitate medium (HP), HP with 3mM AcAc (HP/AcAc), or HP with 3mM 3HB (HP/3HB). Directly after the culturing, cells were used for the [3H]chloroquine accumulation assay. (n = 5). ( B ) Amino acids prevent 3HB-induced v-ATPase inhibition in cardiomyocytes: aRCMs were incubated for 24 h in either LP, HP, BafA, LP supplemented with 1.56 mM Lys, 1.84 mM Leu and 1.36 mM Arg, (LP/4*KLR; for explanation see Section 4.2 ), 3HB (3 mM), 3HB supplemented with the 4*KLR mix (3HB/4*KLR). Directly after the culturing, cells were used for the [3H]chloroquine accumulation assay (n = 5). ( C – F ) 3HB exposure induces v-ATPase disassembly: HL-1 cells were incubated for 24 h in either control (Ctrl) medium, HP medium, or Ctrl medium with 3 mM 3HB (3HB). ( C ) Subcellular fractionation: cytoplasmic fractions (C) and membrane fractions (M) were analyzed by Western blotting of v-ATPase subunits B2 (V1-B2) and d1 (V0-d1), after which the signals were quantified. For V1-B2, the signal ratio of membrane fraction/cytoplasm in control condition is set at 1.0. For V0-d1, the signal density in the membrane fraction is 1.0. The quantified signals in the other conditions are expressed as multiples. Representative blots are displayed. Caveolin-3 (Cav-3) and GAPDH: loading controls for membrane and cytoplasmic fraction, respectively. (n = 4). ( D – F ) Co-Immunoprecipitation (Co-IP) of V1, V0, and mTORC1. ( D ) IP with V1-B2, ( E ) IP with V0-d1. ( F ) IP with mTORC1. Immunoprecipitates were blotted with antibodies against mTORC1, V0-a2, V0-d1, and V1-B2, after which the signals were quantified. Representative Western blots are displayed. (n = 3). Bar values are means ± SEM. * p

    Article Snippet: At day 1 of differentiation, Essential 8 medium was removed and replaced with cardiomyocyte medium a (Thermofisher Scientific, Miami, FL, USA).

    Techniques: Activation Assay, Activity Assay, Cell Culture, Inhibition, Incubation, Fractionation, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay

    Chronic 3HB exposure induces contractile dysfunction in cardiomyocytes. ARCMs were cultured for 24 h under various conditions, being low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB) or 3 mM acetoacetate (AcAc), and LP with 3HB in the presence of 4*KLR (3HB/4*KLR). The contractile parameters ( A ) sarcomere shortening, ( B ) time to peak and ( C ) decay time were deduced during electrostimulation at 1 Hz frequency (n = 4; imaging of 10 cells/measurement condition). Bar values are means ± SEM. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly—Rescue by Specific Amino Acid Supplementation

    doi: 10.3390/ijms232112909

    Figure Lengend Snippet: Chronic 3HB exposure induces contractile dysfunction in cardiomyocytes. ARCMs were cultured for 24 h under various conditions, being low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB) or 3 mM acetoacetate (AcAc), and LP with 3HB in the presence of 4*KLR (3HB/4*KLR). The contractile parameters ( A ) sarcomere shortening, ( B ) time to peak and ( C ) decay time were deduced during electrostimulation at 1 Hz frequency (n = 4; imaging of 10 cells/measurement condition). Bar values are means ± SEM. * p

    Article Snippet: At day 1 of differentiation, Essential 8 medium was removed and replaced with cardiomyocyte medium a (Thermofisher Scientific, Miami, FL, USA).

    Techniques: Cell Culture, Imaging

    Chronic 3HB exposure interferes with insulin signaling in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB), or LP with 3HB in the presence of 4*KLR (3HB/4*KLR). ( A , B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. Thereafter, ( A ) Ser473 phosphorylation of Akt, ( B ) Ser2447 phosphorylation of mTOR, ( C ) Thr 642 phosphorylation of AS160, and ( D ) Ser235/236 phosphorylation of S6 were assessed by Western blotting and quantified. For this, normalization was performed against loading controls. Loading control for the degree of phosphorylation of Akt and AS160 is caveolin-3. Loading control for mTOR and S6 phosphorylation is GAPDHs. Representative blots and corresponding loading controls are displayed (n = 8). Bar values are means ± SEM. * p

    Journal: International Journal of Molecular Sciences

    Article Title: Ketone Body Exposure of Cardiomyocytes Impairs Insulin Sensitivity and Contractile Function through Vacuolar-Type H+-ATPase Disassembly—Rescue by Specific Amino Acid Supplementation

    doi: 10.3390/ijms232112909

    Figure Lengend Snippet: Chronic 3HB exposure interferes with insulin signaling in cardiomyocytes. ARCMs were cultured for 24 h in either low palmitate medium (LP; control condition), high palmitate medium (HP), LP with 3 mM 3HB (3HB), or LP with 3HB in the presence of 4*KLR (3HB/4*KLR). ( A , B ) After 24 h, cells were short-term (30 min) incubated without/with 100 nM insulin. Thereafter, ( A ) Ser473 phosphorylation of Akt, ( B ) Ser2447 phosphorylation of mTOR, ( C ) Thr 642 phosphorylation of AS160, and ( D ) Ser235/236 phosphorylation of S6 were assessed by Western blotting and quantified. For this, normalization was performed against loading controls. Loading control for the degree of phosphorylation of Akt and AS160 is caveolin-3. Loading control for mTOR and S6 phosphorylation is GAPDHs. Representative blots and corresponding loading controls are displayed (n = 8). Bar values are means ± SEM. * p

    Article Snippet: At day 1 of differentiation, Essential 8 medium was removed and replaced with cardiomyocyte medium a (Thermofisher Scientific, Miami, FL, USA).

    Techniques: Cell Culture, Incubation, Western Blot

    SR-iPSCs generate cells of the three germ layers (A–C) Representative immunostainings for markers of the three germ layers upon differentiation of SR-iPSCs towards endoderm (A), cardiomyocytes (mesoderm, B), and neural precursors (ectoderm, C). (D) Negative control: to confirm the specificity of signal, immunostaining of neural precursors was performed excluding primary antibodies. Cells were stained with secondary antibody only. For imaging, the same exposure times as applied in panel C were used. Scale bars: 200 μm (A, C) and 100 μm (B, D).

    Journal: iScience

    Article Title: Induced pluripotent stem cells and cerebral organoids from the critically endangered Sumatran rhinoceros

    doi: 10.1016/j.isci.2022.105414

    Figure Lengend Snippet: SR-iPSCs generate cells of the three germ layers (A–C) Representative immunostainings for markers of the three germ layers upon differentiation of SR-iPSCs towards endoderm (A), cardiomyocytes (mesoderm, B), and neural precursors (ectoderm, C). (D) Negative control: to confirm the specificity of signal, immunostaining of neural precursors was performed excluding primary antibodies. Cells were stained with secondary antibody only. For imaging, the same exposure times as applied in panel C were used. Scale bars: 200 μm (A, C) and 100 μm (B, D).

    Article Snippet: From now on, Cardiomyocyte Maintenance Medium was changed every other day.

    Techniques: Negative Control, Immunostaining, Staining, Imaging