glucose glutamax  (Thermo Fisher)


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    Thermo Fisher glucose glutamax
    Glucose Glutamax, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dmem
    Effect of ammonia. ( a ) Infectious titer of the SRS11.EFS.IL2RG.pre* (GALV-pseudotyped) vector (Avg±s.d.; n =2; ns) transfected in <t>DMEM</t> using 10-layer CellSTACKS or X-Vivo 10 (≤3 months old) in roller bottles supplemented with Fibra-Cel. ( b ) Relative amount of IL2RG vector generated using 3-month- or 9-month-old X-Vivo 10 medium (Avg±s.d.; n =4; ** P =0.01). ( c ) Level of ammonia in X-Vivo 10 medium ranging from 3 to 10 months old as determined on a Roche Diagnostics Hitachi 917 Analyzer. Control is non-ammoniagenic DMEM <t>10564</t> (Invitrogen) supplemented with 10% FBS (Avg±s.d., n =2 for 3-month- and 10-month-old samples, *** P =0.001). Dotted line indicates the published 300 μM level of ammonia toxicity in vitro . ( d ) Infectious titer of the SERS11.GFP.pre* (GALV-pseudotyped) vector generated in 10-month-old X-Vivo 10 medium with or without supplementation with 2 mM glutamine (Avg±s.d., n =2 for Control, n =4 for group supplemented; ns). ( e ) Relative amount of IL2RG vector generated in DMEM medium spiked with increasing concentrations of ammonium chloride (Avg±s.d., n =3; * P
    Dmem, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cardiomyocyte medium
    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: <t>cardiomyocyte</t> nuclei. Data are mean ± SD, **: p
    Cardiomyocyte Medium, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Effect of ammonia. ( a ) Infectious titer of the SRS11.EFS.IL2RG.pre* (GALV-pseudotyped) vector (Avg±s.d.; n =2; ns) transfected in DMEM using 10-layer CellSTACKS or X-Vivo 10 (≤3 months old) in roller bottles supplemented with Fibra-Cel. ( b ) Relative amount of IL2RG vector generated using 3-month- or 9-month-old X-Vivo 10 medium (Avg±s.d.; n =4; ** P =0.01). ( c ) Level of ammonia in X-Vivo 10 medium ranging from 3 to 10 months old as determined on a Roche Diagnostics Hitachi 917 Analyzer. Control is non-ammoniagenic DMEM 10564 (Invitrogen) supplemented with 10% FBS (Avg±s.d., n =2 for 3-month- and 10-month-old samples, *** P =0.001). Dotted line indicates the published 300 μM level of ammonia toxicity in vitro . ( d ) Infectious titer of the SERS11.GFP.pre* (GALV-pseudotyped) vector generated in 10-month-old X-Vivo 10 medium with or without supplementation with 2 mM glutamine (Avg±s.d., n =2 for Control, n =4 for group supplemented; ns). ( e ) Relative amount of IL2RG vector generated in DMEM medium spiked with increasing concentrations of ammonium chloride (Avg±s.d., n =3; * P

    Journal: Gene therapy

    Article Title: Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency

    doi: 10.1038/gt.2012.37

    Figure Lengend Snippet: Effect of ammonia. ( a ) Infectious titer of the SRS11.EFS.IL2RG.pre* (GALV-pseudotyped) vector (Avg±s.d.; n =2; ns) transfected in DMEM using 10-layer CellSTACKS or X-Vivo 10 (≤3 months old) in roller bottles supplemented with Fibra-Cel. ( b ) Relative amount of IL2RG vector generated using 3-month- or 9-month-old X-Vivo 10 medium (Avg±s.d.; n =4; ** P =0.01). ( c ) Level of ammonia in X-Vivo 10 medium ranging from 3 to 10 months old as determined on a Roche Diagnostics Hitachi 917 Analyzer. Control is non-ammoniagenic DMEM 10564 (Invitrogen) supplemented with 10% FBS (Avg±s.d., n =2 for 3-month- and 10-month-old samples, *** P =0.001). Dotted line indicates the published 300 μM level of ammonia toxicity in vitro . ( d ) Infectious titer of the SERS11.GFP.pre* (GALV-pseudotyped) vector generated in 10-month-old X-Vivo 10 medium with or without supplementation with 2 mM glutamine (Avg±s.d., n =2 for Control, n =4 for group supplemented; ns). ( e ) Relative amount of IL2RG vector generated in DMEM medium spiked with increasing concentrations of ammonium chloride (Avg±s.d., n =3; * P

    Article Snippet: Human embryonic kidney-293 cell-derived 293T cells (No. CRL-11268; ATCC, Manassas, VA, USA) were thawed and cultured in DMEM (10564 with GlutaMax; Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS and 1 mM Sodium Pyruvate (Invitrogen).

    Techniques: Plasmid Preparation, Transfection, Generated, In Vitro

    SP does not change extracellular or intracellular zinc levels in DRG cultures. (A) Nominally zinc-free media contains micromolar levels of zinc. Atomic absorption spectroscopy measurements of total zinc in experimental media (as indicated); left eight bars correspond to the standard solutions of ZnCl 2 in de-ionized water. Bars in the middle correspond to the levels of zinc in experimental media: DMEM is a DRG culture medium tested either on its own or collected from a DRG culture plate (“DMEM” and “DMEM+DRG”); EC is an extracellular solution used in patch-clamp and imaging experiments tested either on its own or collected from a DRG culture plate (“EC” and “EC+DRG”). Two bars on the right correspond to the experiments where zinc content has been analyzed in the EC of DRG cultures treated with 1 μ M S9SP for 2 or 10 min (as indicated). (B) SP does not produce detectable rises in intracellular zinc levels in DRG neurons. Fluorescence imaging of cultured small-diameter DRG neurons loaded with zinc fluorophore, FluoZin ™ -3AM. Example time course of the effect of S9SP (1 μ M ), ZnCl 2 (1.5 m M ) and zinc ionophore, ZnPy (10 μ M ) on FluoZin-3 fluorescence. Corresponding images of the culture under basal conditions and after the application of ZnPy (as indicated) are shown on the right . (C) Increase in FluoZin-3 fluorescence induced by 10 μ M ZnPy is completely reversed by 20 μ M zinc chelator, TPEN but is unaffected by 1 m M DTT. (D, E) Summarize the experiments presented in (B, C), respectively. (D) Asterisks indicate significant difference from the basal fluorescence with *** p

    Journal: Antioxidants & Redox Signaling

    Article Title: Redox-Dependent Modulation of T-Type Ca2+ Channels in Sensory Neurons Contributes to Acute Anti-Nociceptive Effect of Substance P

    doi: 10.1089/ars.2015.6560

    Figure Lengend Snippet: SP does not change extracellular or intracellular zinc levels in DRG cultures. (A) Nominally zinc-free media contains micromolar levels of zinc. Atomic absorption spectroscopy measurements of total zinc in experimental media (as indicated); left eight bars correspond to the standard solutions of ZnCl 2 in de-ionized water. Bars in the middle correspond to the levels of zinc in experimental media: DMEM is a DRG culture medium tested either on its own or collected from a DRG culture plate (“DMEM” and “DMEM+DRG”); EC is an extracellular solution used in patch-clamp and imaging experiments tested either on its own or collected from a DRG culture plate (“EC” and “EC+DRG”). Two bars on the right correspond to the experiments where zinc content has been analyzed in the EC of DRG cultures treated with 1 μ M S9SP for 2 or 10 min (as indicated). (B) SP does not produce detectable rises in intracellular zinc levels in DRG neurons. Fluorescence imaging of cultured small-diameter DRG neurons loaded with zinc fluorophore, FluoZin ™ -3AM. Example time course of the effect of S9SP (1 μ M ), ZnCl 2 (1.5 m M ) and zinc ionophore, ZnPy (10 μ M ) on FluoZin-3 fluorescence. Corresponding images of the culture under basal conditions and after the application of ZnPy (as indicated) are shown on the right . (C) Increase in FluoZin-3 fluorescence induced by 10 μ M ZnPy is completely reversed by 20 μ M zinc chelator, TPEN but is unaffected by 1 m M DTT. (D, E) Summarize the experiments presented in (B, C), respectively. (D) Asterisks indicate significant difference from the basal fluorescence with *** p

    Article Snippet: siRNA gene silencing To knock down CaV 3.2, DRGs were dissociated as described ( ); immediately after dissociation, DRG pellets were resuspended in 900 μl DMEM (without GlutaMax I, serum or antibiotics), to which a mixture of 9 μl Lipofectamine RNAiMAX (Invitrogen) and either a mix of three anti-Cacna1h Stealth siRNAs (RSS350285, RSS350286, RSS350287; Life Technologies; 200 nM each) or a scrambled control oligo (Stealth RNAi™ siRNA Negative Control; Life Technologies; 200 nM ) were added immediately before resuspension.

    Techniques: Atomic Absorption Spectroscopy, Patch Clamp, Imaging, Fluorescence, Cell Culture

    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

    Journal: Journal of Cardiovascular Development and Disease

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

    doi: 10.3390/jcdd8080087

    Figure Lengend 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

    Article Snippet: Rat ventricular cardiomyocytes were isolated from Sprague Dawley rats on day 3 after birth (P3) as previously described [ ], seeded on 1 mg/mL fibronectin (Sigma-Aldrich, St. Louis, MO, USA)-coated glass coverslips, and cultured in cardiomyocyte medium (DMEM-F12, Glutamax TM-I, 3 mM Na-pyruvate, 0.2% bovine serum albumin (BSA), 0.1 mM ascorbic acid, 0.5% Insulin-Transferrin-Selenium (100×, Life Technologies, Carlsbad, CA, USA), and penicillin/streptomycin (100 U/mg/mL)) containing 5% horse serum, if not stated otherwise.

    Techniques: 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

    Journal: Journal of Cardiovascular Development and Disease

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

    doi: 10.3390/jcdd8080087

    Figure Lengend 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

    Article Snippet: Rat ventricular cardiomyocytes were isolated from Sprague Dawley rats on day 3 after birth (P3) as previously described [ ], seeded on 1 mg/mL fibronectin (Sigma-Aldrich, St. Louis, MO, USA)-coated glass coverslips, and cultured in cardiomyocyte medium (DMEM-F12, Glutamax TM-I, 3 mM Na-pyruvate, 0.2% bovine serum albumin (BSA), 0.1 mM ascorbic acid, 0.5% Insulin-Transferrin-Selenium (100×, Life Technologies, Carlsbad, CA, USA), and penicillin/streptomycin (100 U/mg/mL)) containing 5% horse serum, if not stated otherwise.

    Techniques: 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

    Journal: Journal of Cardiovascular Development and Disease

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

    doi: 10.3390/jcdd8080087

    Figure Lengend 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

    Article Snippet: Rat ventricular cardiomyocytes were isolated from Sprague Dawley rats on day 3 after birth (P3) as previously described [ ], seeded on 1 mg/mL fibronectin (Sigma-Aldrich, St. Louis, MO, USA)-coated glass coverslips, and cultured in cardiomyocyte medium (DMEM-F12, Glutamax TM-I, 3 mM Na-pyruvate, 0.2% bovine serum albumin (BSA), 0.1 mM ascorbic acid, 0.5% Insulin-Transferrin-Selenium (100×, Life Technologies, Carlsbad, CA, USA), and penicillin/streptomycin (100 U/mg/mL)) containing 5% horse serum, if not stated otherwise.

    Techniques: Immunofluorescence, Transfection, Expressing, Binding Assay

    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

    Journal: eLife

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

    doi: 10.7554/eLife.61669

    Figure Lengend 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

    Article Snippet: After 1.5 hr, non-attached cells, enriched in cardiomyocytes, were collected, centrifuged for 5 min at 330 x g, resuspended in cardiomyocyte medium (DMEM-F12, Glutamax TM-I containing 3 mM Na-pyruvate, 0.2% bovine serum albumin (BSA), 0.1 mM ascorbic acid, 0.5% Insulin-Transferrin-Selenium (100x, Life Technologies) and penicillin/streptomycin (100 U/mg/ml)).

    Techniques: 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

    Journal: eLife

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

    doi: 10.7554/eLife.61669

    Figure Lengend 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

    Article Snippet: After 1.5 hr, non-attached cells, enriched in cardiomyocytes, were collected, centrifuged for 5 min at 330 x g, resuspended in cardiomyocyte medium (DMEM-F12, Glutamax TM-I containing 3 mM Na-pyruvate, 0.2% bovine serum albumin (BSA), 0.1 mM ascorbic acid, 0.5% Insulin-Transferrin-Selenium (100x, Life Technologies) and penicillin/streptomycin (100 U/mg/ml)).

    Techniques: 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

    Journal: eLife

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

    doi: 10.7554/eLife.61669

    Figure Lengend 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

    Article Snippet: After 1.5 hr, non-attached cells, enriched in cardiomyocytes, were collected, centrifuged for 5 min at 330 x g, resuspended in cardiomyocyte medium (DMEM-F12, Glutamax TM-I containing 3 mM Na-pyruvate, 0.2% bovine serum albumin (BSA), 0.1 mM ascorbic acid, 0.5% Insulin-Transferrin-Selenium (100x, Life Technologies) and penicillin/streptomycin (100 U/mg/ml)).

    Techniques: 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.

    Journal: eLife

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

    doi: 10.7554/eLife.61669

    Figure Lengend 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.

    Article Snippet: After 1.5 hr, non-attached cells, enriched in cardiomyocytes, were collected, centrifuged for 5 min at 330 x g, resuspended in cardiomyocyte medium (DMEM-F12, Glutamax TM-I containing 3 mM Na-pyruvate, 0.2% bovine serum albumin (BSA), 0.1 mM ascorbic acid, 0.5% Insulin-Transferrin-Selenium (100x, Life Technologies) and penicillin/streptomycin (100 U/mg/ml)).

    Techniques: 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.

    Journal: eLife

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

    doi: 10.7554/eLife.61669

    Figure Lengend 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.

    Article Snippet: After 1.5 hr, non-attached cells, enriched in cardiomyocytes, were collected, centrifuged for 5 min at 330 x g, resuspended in cardiomyocyte medium (DMEM-F12, Glutamax TM-I containing 3 mM Na-pyruvate, 0.2% bovine serum albumin (BSA), 0.1 mM ascorbic acid, 0.5% Insulin-Transferrin-Selenium (100x, Life Technologies) and penicillin/streptomycin (100 U/mg/ml)).

    Techniques: 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.

    Journal: eLife

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

    doi: 10.7554/eLife.61669

    Figure Lengend 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.

    Article Snippet: After 1.5 hr, non-attached cells, enriched in cardiomyocytes, were collected, centrifuged for 5 min at 330 x g, resuspended in cardiomyocyte medium (DMEM-F12, Glutamax TM-I containing 3 mM Na-pyruvate, 0.2% bovine serum albumin (BSA), 0.1 mM ascorbic acid, 0.5% Insulin-Transferrin-Selenium (100x, Life Technologies) and penicillin/streptomycin (100 U/mg/ml)).

    Techniques: 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

    Journal: eLife

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

    doi: 10.7554/eLife.61669

    Figure Lengend 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

    Article Snippet: After 1.5 hr, non-attached cells, enriched in cardiomyocytes, were collected, centrifuged for 5 min at 330 x g, resuspended in cardiomyocyte medium (DMEM-F12, Glutamax TM-I containing 3 mM Na-pyruvate, 0.2% bovine serum albumin (BSA), 0.1 mM ascorbic acid, 0.5% Insulin-Transferrin-Selenium (100x, Life Technologies) and penicillin/streptomycin (100 U/mg/ml)).

    Techniques: Western Blot, Expressing, Immunostaining