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  • 95
    Thermo Fisher ibmx
    Continuous exposure to albuterol reduces stimulated <t>CFTR</t> activity in a fashion similar to continuous or intermittent dosing with formoterol. wtCFTR + and VX809-corrected F508del-CFTR + CFBE41o- cells were exposed to either continuous or intermittent (1 hour, twice daily) albuterol (10 μM) or the long-acting β2AR-agonist formoterol (LABA; 1 μM) for 72 hours. Cells were mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. ( A ) Representative short-circuit current (I sc ) tracings in wtCFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in B ( n = 4 inserts/condition; circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]). ( C ) Representative I sc tracings in VX809-treated F508del-CFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in D ( n = 4 inserts/condition). All data were normalized to the control or VX809 (no albuterol pretreatment) condition. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (10 μM forskolin/100 μM <t>IBMX;</t> dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR + , 1 μM VX770 for F508del-CFTR + cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). All experiments are representative of studies repeated in duplicate or triplicate with similar results. Data presented represent the mean ± SEM. * P
    Ibmx, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ibmx
    Pharmacological inhibition of K Ca 1.1 channels with <t>paxilline</t> abolished the hyperpolarizing effect of <t>IBMX</t> in human UBSM-isolated cells. A : original current-clamp recording illustrating that 1 μM paxilline abolished STHs and that the subsequent
    Ibmx, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3584 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ibmx  (Tocris)
    95
    Tocris ibmx
    CRIS is a novel target for cAMP. ( A ) Sequence comparison of CNBDs from different proteins. Sequence alignment of CNBDs from mCRIS, cyclic nucleotide-gated channels (bCNGA1, rCNGA4), a hyperpolarization activated and cyclic nucleotide-gated channel (mHCN2), a regulatory subunit from PKA (bPKARI-B), the exchange protein directly-activated by cAMP (hEPAC1), the bacterial catabolite activator protein (CAP), and the ELK1 channel from zebrafish (zELK). Amino acids that have been shown to be essential for ligand binding [1] are highlighted with asterisks. The β strand that functions as an intrinsic ligand in the ELK channels is highlighted in grey. Secondary structure elements are indicated below (β sheets: β 1–8, black arrows; α helices: αA–C, PBC, grey boxes). ( B ) M4T model of the presumed CNBD of mCRIS in the presence of cAMP. ( C ) Close-up view of the CNBD model of mCRIS indicating important interactions of side chain and backbone atoms with cAMP. ( D–G ) Analysis of cAMP binding using FRET. ( D ) Model demonstrating that binding of cAMP changes the conformation of the CNBD resulting in a change in FRET. ( E ) Representative traces for the change in cerulean (blue) and citrine (yellow) emission during perfusion of cit-mCNBD-cer expressing CHO cells with 3 mM 8-Br-cAMP. Arrow indicates start of perfusion. ( F ) Average change in FRET (normalized emission ratio cerulean/FRET-citrine) during perfusion of cit-mCNBD-cer expressing cells with 3 mM 8-Br-cAMP (CNBD 8-Br-cAMP), 40 µM <t>NKH477/100</t> µM <t>IBMX</t> (CNBD NKH/IBMX), 3 mM 8-Br-cGMP (CNBD 8-Br-cGMP), and cit-mCNBD-R288Q-cer expressing cells with 3 mM 8-Br-cAMP (CNBD-RQ 8-Br-cAMP), and 40 µM NKH477/100 µM IBMX (CNBD-RQ NKH/IBMX). Arrow indicates start of perfusion. ( G ) Average change in FRET after 10 min of perfusion (mean ± s.d., black; 95% confident interval, dotted, grey). N numbers and p values are indicated.
    Ibmx, supplied by Tocris, used in various techniques. Bioz Stars score: 95/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM 3 isobutyl 1 methylxanthine
    Kibizu concentrated liquid suppressed accumulation of lipid droplets in 3T3-L1 cells. 3T3-L1 cells were seeded onto a 96-well plate; transferred to Dulbecco’s Modified Eagle Medium (DMEM) containing 10 % FBS, 0.5 mM <t>3-isobutyl-1-methylxanthine,</t>
    3 Isobutyl 1 Methylxanthine, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore isobutyl methyl xanthine ibmx
    BAY 41-2272 (10 µM) and time-dependent increases in cGMP levels in unchallenged WKY washed platelets. Upper panel: with or without <t>IBMX</t> (0.5 mM). Lower panel: with or without <t>ODQ</t> (5 µM). Data are shown as mean ± SEM. N = 4, number
    Isobutyl Methyl Xanthine Ibmx, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Cayman Chemical ibmx
    Augmentation of GSIS over baseline from HIT-T15 cells in static incubations containing 11.1 mM glucose over 2 h. A series of increasing concentrations of phosphodiesterase inhibitors was compared. The first evidence for the augmentation of GSIS occurred at a concentration of 20 µM for all drugs, and maximum responses occurred at 100 µM, except for <t>milrinone</t> and <t>3-isobutyl-1-methylxanthine</t> <t>(IBMX),</t> whose augmentation effects continued to rise. Data = mean ± SE; experiments performed in triplicate.
    Ibmx, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 92/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem ibmx
    Acid secretory capacity declines in parallel with the decline in parietal cell number. Time course of acid secretory rates in isolated gastric mucosae of (a) 8‐day‐, (b) 1‐month‐ and (c) 6‐month‐old Car9 −/− and WT mice. (d) Basal and (e) maximal acid secretory rates after stimulation with <t>forskolin</t> ( FSK ) and <t>IBMX</t> were significantly lower in Car9 −/− mice than in WT up from 3 months of age. The decline in acid secretory capacity in the mucosa of Car9 −/− mice is paralleled by the decline in parietal cell numbers (see Fig. 2 ). n = 6–7, * P
    Ibmx, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore isobutyl 1 methylxanthine ibmx
    Expansion and trilineage differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). (A) Cell counts, (B) population doublings per day, and (C) cumulative population doublings at each passage during expansion of BMSCs under normoxia or hypoxia. Data points represent mean ± standard error of the mean of cells from six donors, and P values are listed. (D) Osteogenic differentiation of BMSCs verified with Alizarin Red S staining following expansion under hypoxia and monolayer culture within medium containing β-glycerophosphate, dexamethasone, and fetal bovine serum. (E) Adipogenic differentiation of BMSCs verified with Oil Red O staining following expansion under hypoxia and monolayer culture within medium containing <t>isobutyl-1-methylxanthine</t> <t>(IBMX),</t> indomethacin, dexamethasone, and fetal bovine serum. (F) Chondrogenic differentiation of BMSCs verified by safranin O staining following expansion under hypoxia and culture performed in pellets (left) or scaffolds composed of collagen (middle) or hyaluronic acid (right) submersed in a defined serum-free chondrogenic medium containing transforming growth factor-beta 3 and dexamethasone.
    Isobutyl 1 Methylxanthine Ibmx, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Biomol GmbH 3 isobutyl 1 methylxanthine
    Expansion and trilineage differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). (A) Cell counts, (B) population doublings per day, and (C) cumulative population doublings at each passage during expansion of BMSCs under normoxia or hypoxia. Data points represent mean ± standard error of the mean of cells from six donors, and P values are listed. (D) Osteogenic differentiation of BMSCs verified with Alizarin Red S staining following expansion under hypoxia and monolayer culture within medium containing β-glycerophosphate, dexamethasone, and fetal bovine serum. (E) Adipogenic differentiation of BMSCs verified with Oil Red O staining following expansion under hypoxia and monolayer culture within medium containing <t>isobutyl-1-methylxanthine</t> <t>(IBMX),</t> indomethacin, dexamethasone, and fetal bovine serum. (F) Chondrogenic differentiation of BMSCs verified by safranin O staining following expansion under hypoxia and culture performed in pellets (left) or scaffolds composed of collagen (middle) or hyaluronic acid (right) submersed in a defined serum-free chondrogenic medium containing transforming growth factor-beta 3 and dexamethasone.
    3 Isobutyl 1 Methylxanthine, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Nacalai 3 isobutyl 1 methylxanthine
    Comparison of ASC differentiation to adipocytes. Comparison of adipocyte differentiation between the WT ( Pin1 +/+ ; p53 -/- ) ASC and Pin1-KO ( Pin1 -/- ; p53 -/- ) ASC, and Pin1-KO ASC rescued with the lentiviral Pin1 cDNA. The wild type ASC, the Pin1-KO ASC- infected with Mock and the Pin1-KO ASC- infected with lentiviral Pin1 cDNA were cultured in DMEM containing 0.5 mM <t>3-isobutyl-1-methylxanthine,</t> 1 μM dexamethasone, and 1.7 μM insulin for 0–16 days. ASCs were treated with 4% paraformaldehyde and 60% 2-propanol, and then stained with Oil Red O. The images of the oil red O-stained cells on 0, 4, 7, 10, 13, and 16 days after treatment with the differentiation reagent are shown.
    3 Isobutyl 1 Methylxanthine, supplied by Nacalai, used in various techniques. Bioz Stars score: 90/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Merck KGaA ibmx
    Comparison of ASC differentiation to adipocytes. Comparison of adipocyte differentiation between the WT ( Pin1 +/+ ; p53 -/- ) ASC and Pin1-KO ( Pin1 -/- ; p53 -/- ) ASC, and Pin1-KO ASC rescued with the lentiviral Pin1 cDNA. The wild type ASC, the Pin1-KO ASC- infected with Mock and the Pin1-KO ASC- infected with lentiviral Pin1 cDNA were cultured in DMEM containing 0.5 mM <t>3-isobutyl-1-methylxanthine,</t> 1 μM dexamethasone, and 1.7 μM insulin for 0–16 days. ASCs were treated with 4% paraformaldehyde and 60% 2-propanol, and then stained with Oil Red O. The images of the oil red O-stained cells on 0, 4, 7, 10, 13, and 16 days after treatment with the differentiation reagent are shown.
    Ibmx, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 91/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Applichem ibmx
    Effects of late I Na induction on cellular cAMP levels in atrial CMs of mice. ( A–D ) YFP/CFP tracings in CMs of EPAC-camps transgenic mice upon ATX-II (0.5 nmol/L) and subsequent isoproterenol treatment (100 nmol/L). CMs were <t>preincubated</t> with a ( B ) PDE inhibitor <t>IBMX</t> (100 µmol/L), ( C ) an inhibitor of AC 5/6 (NKY80, 10 µmol/L), ( D ) an inhibitor of reverse-mode NCX KBR (0.1 µmol/L), or ( A ) no inhibitor. ( E ) Quantification of response of YFP/CFP ratio ( n mice/cells = 10/39 vs. 3/8 vs. 3/10 vs. 3/11). *significant vs. steady-state before ATX-II; # significant vs. YFP/CFP ratio upon ATX-II in the control group. * / # P
    Ibmx, supplied by Applichem, used in various techniques. Bioz Stars score: 93/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ibmx free m16 medium
    Effects of late I Na induction on cellular cAMP levels in atrial CMs of mice. ( A–D ) YFP/CFP tracings in CMs of EPAC-camps transgenic mice upon ATX-II (0.5 nmol/L) and subsequent isoproterenol treatment (100 nmol/L). CMs were <t>preincubated</t> with a ( B ) PDE inhibitor <t>IBMX</t> (100 µmol/L), ( C ) an inhibitor of AC 5/6 (NKY80, 10 µmol/L), ( D ) an inhibitor of reverse-mode NCX KBR (0.1 µmol/L), or ( A ) no inhibitor. ( E ) Quantification of response of YFP/CFP ratio ( n mice/cells = 10/39 vs. 3/8 vs. 3/10 vs. 3/11). *significant vs. steady-state before ATX-II; # significant vs. YFP/CFP ratio upon ATX-II in the control group. * / # P
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    90
    Santa Cruz Biotechnology ibmx
    Investigation of phosphodiesterase regulation of β 3 -AR/cGMP in adult cardiomyocytes. Representative FRET response curves of control (grey line) and failing (black line) cardiomyocytes following whole cell treatment with isoproterenol (100 nmol/L) followed by the PDE1 blocker vinpocetine (VINPO, 10 µmol/L) ( A ), the PDE2 inhibitor EHNA (10 µmol/L) ( B ), the PDE3 inhibitor <t>cilostamide</t> (CILO, 10 µmol/L) ( C ) and the PDE5 inhibitor tadalafil (TAD, 100 nmol /L) ( D ) followed by the non-specific PDE blocker <t>IBMX</t> (100 µmol/L). The scatter plot/histograms present whole cell cGMP-FRET responses evoked by PDE inhibition further to the isoproterenol responses in % from ( A–D ) ( E ) Error bars represent standard error of the mean. Numbers of cells/hearts are shown below the bars. Statistical significance was calculated via mixed ANOVA followed by χ2-test: No statistically significant differences between control and failing conditions for any PDE could be detected, only tendencies to increased responses for PDE2, PDE3 and PDE5 inhibitors. FRET microscopy data - 'whole cell' analysis PDEs.
    Ibmx, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Continuous exposure to albuterol reduces stimulated CFTR activity in a fashion similar to continuous or intermittent dosing with formoterol. wtCFTR + and VX809-corrected F508del-CFTR + CFBE41o- cells were exposed to either continuous or intermittent (1 hour, twice daily) albuterol (10 μM) or the long-acting β2AR-agonist formoterol (LABA; 1 μM) for 72 hours. Cells were mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. ( A ) Representative short-circuit current (I sc ) tracings in wtCFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in B ( n = 4 inserts/condition; circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]). ( C ) Representative I sc tracings in VX809-treated F508del-CFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in D ( n = 4 inserts/condition). All data were normalized to the control or VX809 (no albuterol pretreatment) condition. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (10 μM forskolin/100 μM IBMX; dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR + , 1 μM VX770 for F508del-CFTR + cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). All experiments are representative of studies repeated in duplicate or triplicate with similar results. Data presented represent the mean ± SEM. * P

    Journal: JCI Insight

    Article Title: Chronic β2AR stimulation limits CFTR activation in human airway epithelia

    doi: 10.1172/jci.insight.93029

    Figure Lengend Snippet: Continuous exposure to albuterol reduces stimulated CFTR activity in a fashion similar to continuous or intermittent dosing with formoterol. wtCFTR + and VX809-corrected F508del-CFTR + CFBE41o- cells were exposed to either continuous or intermittent (1 hour, twice daily) albuterol (10 μM) or the long-acting β2AR-agonist formoterol (LABA; 1 μM) for 72 hours. Cells were mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. ( A ) Representative short-circuit current (I sc ) tracings in wtCFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in B ( n = 4 inserts/condition; circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]). ( C ) Representative I sc tracings in VX809-treated F508del-CFTR + CFBE41o- cells exposed for 72 hours to either continuous (C) or intermittent (I) albuterol or formoterol; aggregate data are presented in D ( n = 4 inserts/condition). All data were normalized to the control or VX809 (no albuterol pretreatment) condition. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (10 μM forskolin/100 μM IBMX; dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR + , 1 μM VX770 for F508del-CFTR + cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). All experiments are representative of studies repeated in duplicate or triplicate with similar results. Data presented represent the mean ± SEM. * P

    Article Snippet: CFTR was activated with apical/basolateral forskolin (10 μM; Sigma-Aldrich) and IBMX (100 μM; Acros Organics) and then potentiated with apical genistein (50 μM, Sigma-Aldrich, wtCFTR) or VX770 (1 μM, Selleck Chemicals, F508del/F508del).

    Techniques: Activity Assay, Inhibition

    Chronic albuterol-induced CFTR impairment is rescued with application of exogenous cAMP. wtCFTR + and VX809-corrected F508del-CFTR + CFBE41o- cells were exposed to albuterol and/or VX809 in media for 72 hours, then mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. In contrast to previously demonstrated experiments, cell-permeant 8-bromoadenosine cAMP (8-Br cAMP) was used to stimulate CFTR function to bypass cellular mechanisms of cAMP generation. In wtCFTR + cells (representative short-circuit current [I sc ] tracing in A , aggregate data in B ; n = 4 inserts/condition. Circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]), maximal stimulation with 8-Br cAMP in place of forskolin/IBMX rescues the previously demonstrated albuterol-induced CFTR dysfunction. Stimulation with increasing doses of 8-Br cAMP ( C , n = 3 inserts/data point) revealed a small increase in CFTR function in albuterol-pretreated cells at low stimulation doses. Squares, control conditions; triangles, albuterol pretreated. Similarly, in VX809-corrected F508del-CFTR + cells (representative I sc tracing in D , aggregate data in E ; n = 4 inserts/condition), maximal stimulation with 8-Br cAMP rescues albuterol-induced CFTR dysfunction. Unlike wtCFTR + cells, stimulation with increasing doses of 8-Br cAMP demonstrated no difference in VX809-corrected F508del-CFTR function following albuterol pretreatment at any stimulation dose ( F , n = 3 inserts/data point. All indicators of statistical significance are comparisons with untreated cells; no difference was noted between VX809-corrected cells with or without albuterol). Circles, untreated; squares, VX809 alone; triangles, VX809 + albuterol. All studies are internally normalized as indicated to allow for comparisons. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (100 μM 8-Br cAMP; dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR + ; 1 μM VX770 for F508del-CFTR + cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). Both experiments are representative of studies repeated in duplicate with similar results. Data presented represent the mean ± SEM. * P

    Journal: JCI Insight

    Article Title: Chronic β2AR stimulation limits CFTR activation in human airway epithelia

    doi: 10.1172/jci.insight.93029

    Figure Lengend Snippet: Chronic albuterol-induced CFTR impairment is rescued with application of exogenous cAMP. wtCFTR + and VX809-corrected F508del-CFTR + CFBE41o- cells were exposed to albuterol and/or VX809 in media for 72 hours, then mounted in Ussing chambers and CFTR function was assessed under voltage clamp conditions. In contrast to previously demonstrated experiments, cell-permeant 8-bromoadenosine cAMP (8-Br cAMP) was used to stimulate CFTR function to bypass cellular mechanisms of cAMP generation. In wtCFTR + cells (representative short-circuit current [I sc ] tracing in A , aggregate data in B ; n = 4 inserts/condition. Circles represent total CFTR activity [cAMP + genistein], diamonds represent inhibited CFTR currents [Inh172]), maximal stimulation with 8-Br cAMP in place of forskolin/IBMX rescues the previously demonstrated albuterol-induced CFTR dysfunction. Stimulation with increasing doses of 8-Br cAMP ( C , n = 3 inserts/data point) revealed a small increase in CFTR function in albuterol-pretreated cells at low stimulation doses. Squares, control conditions; triangles, albuterol pretreated. Similarly, in VX809-corrected F508del-CFTR + cells (representative I sc tracing in D , aggregate data in E ; n = 4 inserts/condition), maximal stimulation with 8-Br cAMP rescues albuterol-induced CFTR dysfunction. Unlike wtCFTR + cells, stimulation with increasing doses of 8-Br cAMP demonstrated no difference in VX809-corrected F508del-CFTR function following albuterol pretreatment at any stimulation dose ( F , n = 3 inserts/data point. All indicators of statistical significance are comparisons with untreated cells; no difference was noted between VX809-corrected cells with or without albuterol). Circles, untreated; squares, VX809 alone; triangles, VX809 + albuterol. All studies are internally normalized as indicated to allow for comparisons. Stimulation protocol was as follows: amiloride (100 μM, not shown), cAMP (100 μM 8-Br cAMP; dark gray bars), CFTR potentiator (50 μM genistein for wtCFTR + ; 1 μM VX770 for F508del-CFTR + cells; light gray bars), and CFTR inhibition (10 μM Inh172; white bars). Both experiments are representative of studies repeated in duplicate with similar results. Data presented represent the mean ± SEM. * P

    Article Snippet: CFTR was activated with apical/basolateral forskolin (10 μM; Sigma-Aldrich) and IBMX (100 μM; Acros Organics) and then potentiated with apical genistein (50 μM, Sigma-Aldrich, wtCFTR) or VX770 (1 μM, Selleck Chemicals, F508del/F508del).

    Techniques: Activity Assay, Inhibition

    Chronic albuterol exposure reduces forskolin/IBMX–stimulated generation of cAMP in wtCFTR + and F508del-CFTR + CFBE41o- cells. wtCFTR + and F508del-CFTR + CFBE41o- cells were pretreated with albuterol (10 μM) for 72 hours, then stimulated to produce cAMP with 10 μM forskolin/100 μM IBMX (10 minutes). cAMP levels were then analyzed by colorimetric ELISA. wtCFTR + ( A , n = 6) and F508del-CFTR + ( B , n = 6) CFBE41o- cells produce minimal cAMP in the absence of either pretreatment or stimulation (open circles). Stimulation with 10 μM forskolin/100 μM IBMX in untreated cells leads to cAMP production (open squares), which is reduced by more than 60% in cells pretreated for 72 hours with albuterol (open triangles). All studies are internally normalized to control conditions to allow for comparisons. Both experiments are representative of studies repeated in duplicate with similar results. Data presented represent the mean ± SEM. n = 6 wells per condition. **** P

    Journal: JCI Insight

    Article Title: Chronic β2AR stimulation limits CFTR activation in human airway epithelia

    doi: 10.1172/jci.insight.93029

    Figure Lengend Snippet: Chronic albuterol exposure reduces forskolin/IBMX–stimulated generation of cAMP in wtCFTR + and F508del-CFTR + CFBE41o- cells. wtCFTR + and F508del-CFTR + CFBE41o- cells were pretreated with albuterol (10 μM) for 72 hours, then stimulated to produce cAMP with 10 μM forskolin/100 μM IBMX (10 minutes). cAMP levels were then analyzed by colorimetric ELISA. wtCFTR + ( A , n = 6) and F508del-CFTR + ( B , n = 6) CFBE41o- cells produce minimal cAMP in the absence of either pretreatment or stimulation (open circles). Stimulation with 10 μM forskolin/100 μM IBMX in untreated cells leads to cAMP production (open squares), which is reduced by more than 60% in cells pretreated for 72 hours with albuterol (open triangles). All studies are internally normalized to control conditions to allow for comparisons. Both experiments are representative of studies repeated in duplicate with similar results. Data presented represent the mean ± SEM. n = 6 wells per condition. **** P

    Article Snippet: CFTR was activated with apical/basolateral forskolin (10 μM; Sigma-Aldrich) and IBMX (100 μM; Acros Organics) and then potentiated with apical genistein (50 μM, Sigma-Aldrich, wtCFTR) or VX770 (1 μM, Selleck Chemicals, F508del/F508del).

    Techniques: Enzyme-linked Immunosorbent Assay

    Pharmacological inhibition of K Ca 1.1 channels with paxilline abolished the hyperpolarizing effect of IBMX in human UBSM-isolated cells. A : original current-clamp recording illustrating that 1 μM paxilline abolished STHs and that the subsequent

    Journal: American Journal of Physiology - Renal Physiology

    Article Title: Constitutively active phosphodiesterase activity regulates urinary bladder smooth muscle function: critical role of KCa1.1 channel

    doi: 10.1152/ajprenal.00351.2012

    Figure Lengend Snippet: Pharmacological inhibition of K Ca 1.1 channels with paxilline abolished the hyperpolarizing effect of IBMX in human UBSM-isolated cells. A : original current-clamp recording illustrating that 1 μM paxilline abolished STHs and that the subsequent

    Article Snippet: IBMX, paxilline, fura 2-AM, collagenase (type II), and TTX (in citrate buffer) were purchased from Sigma (St. Louis, MO).

    Techniques: Inhibition, Isolation

    LCoR inhibits the transcriptional activity of C/EBPβ and mitotic clonal expansion. A–C , HEK 293T cells were transiently transfected with a luciferase reporter plasmid containing PPAR γ 2 promoter (−602 to +52) ( PPAR γ 2-luc ) with or without a C/EBPβ construct in the absence or presence of increasing amounts of LCoR ( A ), siRNAs targeting LCoR ( B ), or full-length or truncated LCoR as indicated ( C ). The luciferase activity was measured and is shown as relative luciferase units ( RLU ). D–F , 3T3-L1 cells were infected with retrovirus expressing a PPAR γ 2 promoter (−602 to +52)-driven luciferase reporter with or without retrovirus expressing F-LCoR ( D ), shLCoR ( E ), or truncated LCoR ( F ) as indicated. The luciferase activities were analyzed before or 48 h after differentiation induction using insulin, dexamethasone, and 3-isobutyl-1-methylxanthine ( IDM ) and shown as relative luciferase units ( RLU ). G and H , representative image ( G ) and percentages of BrdU-positive cells ( H ) are shown. 18 h after adipogenic induction, 3T3-L1 cells stably overexpressing F-LCoR or an empty vector (pMSCV) were labeled with BrdU for 2 h and then stained with DAPI. The fluorescence of BrdU ( green ) and DAPI ( blue ) was detected with a fluorescence microscope. I–L , 3T3-L1 cells stably overexpressing F-LCoR or pMSCV were harvested at the indicated times after adipogenic induction. The mRNA levels of CCND2 ( I ), CCNE1 ( J ), Gins1 ( K ), and Mcm3 ( L ) were measured by qPCR analysis. M , the efficiency of the knockdown of C/EBPβ in 3T3-L1 cells was determined by Western blotting. 3T3-L1 preadipocytes were infected with a retroviral vector containing shC/EBPβ or shLacZ as a control. N–Q , 3T3-L1 cells infected with shC/EBPβ or shLacZ were harvested at the indicated times after adipogenic induction. The mRNA levels of CCND2 ( N ), CCNE1 ( O ), Gins1 ( P ), and Mcm3 ( Q ) were measured by qPCR analysis. Blots shown here are representative of at least three independent experiments. Data are represented as mean ± S.D. ( n = 3–4). Error bars represent S.D. *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Ligand-dependent corepressor (LCoR) represses the transcription factor C/EBPβ during early adipocyte differentiation

    doi: 10.1074/jbc.M117.793984

    Figure Lengend Snippet: LCoR inhibits the transcriptional activity of C/EBPβ and mitotic clonal expansion. A–C , HEK 293T cells were transiently transfected with a luciferase reporter plasmid containing PPAR γ 2 promoter (−602 to +52) ( PPAR γ 2-luc ) with or without a C/EBPβ construct in the absence or presence of increasing amounts of LCoR ( A ), siRNAs targeting LCoR ( B ), or full-length or truncated LCoR as indicated ( C ). The luciferase activity was measured and is shown as relative luciferase units ( RLU ). D–F , 3T3-L1 cells were infected with retrovirus expressing a PPAR γ 2 promoter (−602 to +52)-driven luciferase reporter with or without retrovirus expressing F-LCoR ( D ), shLCoR ( E ), or truncated LCoR ( F ) as indicated. The luciferase activities were analyzed before or 48 h after differentiation induction using insulin, dexamethasone, and 3-isobutyl-1-methylxanthine ( IDM ) and shown as relative luciferase units ( RLU ). G and H , representative image ( G ) and percentages of BrdU-positive cells ( H ) are shown. 18 h after adipogenic induction, 3T3-L1 cells stably overexpressing F-LCoR or an empty vector (pMSCV) were labeled with BrdU for 2 h and then stained with DAPI. The fluorescence of BrdU ( green ) and DAPI ( blue ) was detected with a fluorescence microscope. I–L , 3T3-L1 cells stably overexpressing F-LCoR or pMSCV were harvested at the indicated times after adipogenic induction. The mRNA levels of CCND2 ( I ), CCNE1 ( J ), Gins1 ( K ), and Mcm3 ( L ) were measured by qPCR analysis. M , the efficiency of the knockdown of C/EBPβ in 3T3-L1 cells was determined by Western blotting. 3T3-L1 preadipocytes were infected with a retroviral vector containing shC/EBPβ or shLacZ as a control. N–Q , 3T3-L1 cells infected with shC/EBPβ or shLacZ were harvested at the indicated times after adipogenic induction. The mRNA levels of CCND2 ( N ), CCNE1 ( O ), Gins1 ( P ), and Mcm3 ( Q ) were measured by qPCR analysis. Blots shown here are representative of at least three independent experiments. Data are represented as mean ± S.D. ( n = 3–4). Error bars represent S.D. *, p

    Article Snippet: For adipogenic differentiation, 2-day postconfluent 3T3-L1 preadipocytes were induced by adipogenic differentiation medium containing high-glucose DMEM, 10% FBS, 1 μg/ml insulin (Sigma I1882), 1 μ m dexamethasone (Sigma D1756), and 0.5 m m 3-isobutyl-1-methylxanthine (Sigma I5879) for 2 days.

    Techniques: Activity Assay, Transfection, Luciferase, Plasmid Preparation, Construct, Infection, Expressing, Stable Transfection, Labeling, Staining, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Western Blot

    PTH-induced urinary cAMP, phosphate excretion, and cAMP formation in renal cortical tubule suspensions are impaired in AC6 −/− mice. (A) Bolus application of PTH in WT mice dose-dependently increased fractional urinary P i excretion and urinary cAMP excretion. In contrast to WT mice, the phosphaturic effect and cAMP formation in response to PTH are absent in AC6 −/− mice. (B) Stimulation of cAMP formation by forskolin (Forsk; 10 µ mol/L) or PTH is absent in freshly isolated renal cortical tubule suspensions of AC6 −/− compared with WT mice. Protein concentration was adjusted to 60 µ g/vial. Experiments were performed with phosphodiesterase inhibition (0.5 mmol/L 3-isobutyl-1-methylxanthine; n =6–8/genotype for clearance experiments and n =5/genotype for isolated cortical tubules). bw, body weight. * P

    Journal: Journal of the American Society of Nephrology : JASN

    Article Title: Renal Phosphate Wasting in the Absence of Adenylyl Cyclase 6

    doi: 10.1681/ASN.2013101102

    Figure Lengend Snippet: PTH-induced urinary cAMP, phosphate excretion, and cAMP formation in renal cortical tubule suspensions are impaired in AC6 −/− mice. (A) Bolus application of PTH in WT mice dose-dependently increased fractional urinary P i excretion and urinary cAMP excretion. In contrast to WT mice, the phosphaturic effect and cAMP formation in response to PTH are absent in AC6 −/− mice. (B) Stimulation of cAMP formation by forskolin (Forsk; 10 µ mol/L) or PTH is absent in freshly isolated renal cortical tubule suspensions of AC6 −/− compared with WT mice. Protein concentration was adjusted to 60 µ g/vial. Experiments were performed with phosphodiesterase inhibition (0.5 mmol/L 3-isobutyl-1-methylxanthine; n =6–8/genotype for clearance experiments and n =5/genotype for isolated cortical tubules). bw, body weight. * P

    Article Snippet: Renal cortex was isolated using a modification of the method described by Guder, and cAMP accumulation was measured as described., Tubule suspensions were stimulated with PTH (1 nmol/L to 10 µ mol/L; GenScript), forskolin (10 µ mol/L; Ascent Scientific, Princeton, NJ), or vehicle for 15 minutes at 37°C in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (pretreated for 10 minutes, 0.5 mmol/L; Sigma-Aldrich, St. Louis, MO).

    Techniques: Mouse Assay, Isolation, Protein Concentration, Inhibition

    CRIS is a novel target for cAMP. ( A ) Sequence comparison of CNBDs from different proteins. Sequence alignment of CNBDs from mCRIS, cyclic nucleotide-gated channels (bCNGA1, rCNGA4), a hyperpolarization activated and cyclic nucleotide-gated channel (mHCN2), a regulatory subunit from PKA (bPKARI-B), the exchange protein directly-activated by cAMP (hEPAC1), the bacterial catabolite activator protein (CAP), and the ELK1 channel from zebrafish (zELK). Amino acids that have been shown to be essential for ligand binding [1] are highlighted with asterisks. The β strand that functions as an intrinsic ligand in the ELK channels is highlighted in grey. Secondary structure elements are indicated below (β sheets: β 1–8, black arrows; α helices: αA–C, PBC, grey boxes). ( B ) M4T model of the presumed CNBD of mCRIS in the presence of cAMP. ( C ) Close-up view of the CNBD model of mCRIS indicating important interactions of side chain and backbone atoms with cAMP. ( D–G ) Analysis of cAMP binding using FRET. ( D ) Model demonstrating that binding of cAMP changes the conformation of the CNBD resulting in a change in FRET. ( E ) Representative traces for the change in cerulean (blue) and citrine (yellow) emission during perfusion of cit-mCNBD-cer expressing CHO cells with 3 mM 8-Br-cAMP. Arrow indicates start of perfusion. ( F ) Average change in FRET (normalized emission ratio cerulean/FRET-citrine) during perfusion of cit-mCNBD-cer expressing cells with 3 mM 8-Br-cAMP (CNBD 8-Br-cAMP), 40 µM NKH477/100 µM IBMX (CNBD NKH/IBMX), 3 mM 8-Br-cGMP (CNBD 8-Br-cGMP), and cit-mCNBD-R288Q-cer expressing cells with 3 mM 8-Br-cAMP (CNBD-RQ 8-Br-cAMP), and 40 µM NKH477/100 µM IBMX (CNBD-RQ NKH/IBMX). Arrow indicates start of perfusion. ( G ) Average change in FRET after 10 min of perfusion (mean ± s.d., black; 95% confident interval, dotted, grey). N numbers and p values are indicated.

    Journal: PLoS Genetics

    Article Title: CRIS--A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending

    doi: 10.1371/journal.pgen.1003960

    Figure Lengend Snippet: CRIS is a novel target for cAMP. ( A ) Sequence comparison of CNBDs from different proteins. Sequence alignment of CNBDs from mCRIS, cyclic nucleotide-gated channels (bCNGA1, rCNGA4), a hyperpolarization activated and cyclic nucleotide-gated channel (mHCN2), a regulatory subunit from PKA (bPKARI-B), the exchange protein directly-activated by cAMP (hEPAC1), the bacterial catabolite activator protein (CAP), and the ELK1 channel from zebrafish (zELK). Amino acids that have been shown to be essential for ligand binding [1] are highlighted with asterisks. The β strand that functions as an intrinsic ligand in the ELK channels is highlighted in grey. Secondary structure elements are indicated below (β sheets: β 1–8, black arrows; α helices: αA–C, PBC, grey boxes). ( B ) M4T model of the presumed CNBD of mCRIS in the presence of cAMP. ( C ) Close-up view of the CNBD model of mCRIS indicating important interactions of side chain and backbone atoms with cAMP. ( D–G ) Analysis of cAMP binding using FRET. ( D ) Model demonstrating that binding of cAMP changes the conformation of the CNBD resulting in a change in FRET. ( E ) Representative traces for the change in cerulean (blue) and citrine (yellow) emission during perfusion of cit-mCNBD-cer expressing CHO cells with 3 mM 8-Br-cAMP. Arrow indicates start of perfusion. ( F ) Average change in FRET (normalized emission ratio cerulean/FRET-citrine) during perfusion of cit-mCNBD-cer expressing cells with 3 mM 8-Br-cAMP (CNBD 8-Br-cAMP), 40 µM NKH477/100 µM IBMX (CNBD NKH/IBMX), 3 mM 8-Br-cGMP (CNBD 8-Br-cGMP), and cit-mCNBD-R288Q-cer expressing cells with 3 mM 8-Br-cAMP (CNBD-RQ 8-Br-cAMP), and 40 µM NKH477/100 µM IBMX (CNBD-RQ NKH/IBMX). Arrow indicates start of perfusion. ( G ) Average change in FRET after 10 min of perfusion (mean ± s.d., black; 95% confident interval, dotted, grey). N numbers and p values are indicated.

    Article Snippet: CHO cells expressing the FRET sensors were perfused with buffer (140 mM NaCl, 5.4 mM KCl, 1 mM MgCl2 , 1.8 mM CaCl2 , 5 mM HEPES) with or without 40 µM NKH477, 100 µM IBMX (Tocris), and 3 mM 8-Br-cAMP/cGMP (BioLog) 1 min after starting the recording.

    Techniques: Sequencing, Ligand Binding Assay, Binding Assay, Expressing

    Kibizu concentrated liquid suppressed accumulation of lipid droplets in 3T3-L1 cells. 3T3-L1 cells were seeded onto a 96-well plate; transferred to Dulbecco’s Modified Eagle Medium (DMEM) containing 10 % FBS, 0.5 mM 3-isobutyl-1-methylxanthine,

    Journal: Cytotechnology

    Article Title: Kibizu concentrated liquid suppresses the accumulation of lipid droplets in 3T3-L1 cells

    doi: 10.1007/s10616-015-9849-x

    Figure Lengend Snippet: Kibizu concentrated liquid suppressed accumulation of lipid droplets in 3T3-L1 cells. 3T3-L1 cells were seeded onto a 96-well plate; transferred to Dulbecco’s Modified Eagle Medium (DMEM) containing 10 % FBS, 0.5 mM 3-isobutyl-1-methylxanthine,

    Article Snippet: The preadipocytes were differentiated in DMEM containing 10 % FBS, 0.5 mM 3-isobutyl-1-methylxanthine (Wako, Osaka, Japan), 0.25 μM dexamethasone, and 10 μg/mL insulin for 72 h (Phase I).

    Techniques: Modification

    BAY 41-2272 (10 µM) and time-dependent increases in cGMP levels in unchallenged WKY washed platelets. Upper panel: with or without IBMX (0.5 mM). Lower panel: with or without ODQ (5 µM). Data are shown as mean ± SEM. N = 4, number

    Journal: British Journal of Pharmacology

    Article Title: The anti-aggregating effect of BAY 41-2272, a stimulator of soluble guanylyl cyclase, requires the presence of nitric oxide

    doi: 10.1111/j.1476-5381.2010.00943.x

    Figure Lengend Snippet: BAY 41-2272 (10 µM) and time-dependent increases in cGMP levels in unchallenged WKY washed platelets. Upper panel: with or without IBMX (0.5 mM). Lower panel: with or without ODQ (5 µM). Data are shown as mean ± SEM. N = 4, number

    Article Snippet: Adenosine 5′-diphosphate, ODQ, isobutyl-methyl-xanthine (IBMX), L-NA, hydroxocobalamin, sodium nitroprusside, thrombin (Sigma, La Verpillère, France); nitroglycerin (Merck, Darmstadt, Germany); DEA-NONOate (Alexis Chemical, Coger, Paris France); beraprost (Cayman Chemical, Ann Arbor, MI, USA); collagen (Kordia, Leiden, the Netherlands).

    Techniques:

    Augmentation of GSIS over baseline from HIT-T15 cells in static incubations containing 11.1 mM glucose over 2 h. A series of increasing concentrations of phosphodiesterase inhibitors was compared. The first evidence for the augmentation of GSIS occurred at a concentration of 20 µM for all drugs, and maximum responses occurred at 100 µM, except for milrinone and 3-isobutyl-1-methylxanthine (IBMX), whose augmentation effects continued to rise. Data = mean ± SE; experiments performed in triplicate.

    Journal: Antioxidants

    Article Title: Silymarin Activates c-AMP Phosphodiesterase and Stimulates Insulin Secretion in a Glucose-Dependent Manner in HIT-T15 Cells

    doi: 10.3390/antiox5040047

    Figure Lengend Snippet: Augmentation of GSIS over baseline from HIT-T15 cells in static incubations containing 11.1 mM glucose over 2 h. A series of increasing concentrations of phosphodiesterase inhibitors was compared. The first evidence for the augmentation of GSIS occurred at a concentration of 20 µM for all drugs, and maximum responses occurred at 100 µM, except for milrinone and 3-isobutyl-1-methylxanthine (IBMX), whose augmentation effects continued to rise. Data = mean ± SE; experiments performed in triplicate.

    Article Snippet: Sources of Reagents Silymarin and silibin were obtained from Sigma Aldrich, St. Louis, MO, USA; milrinone, rolipram, and IBMX were obtained from Cayman, Ann Arbor, MI, USA.

    Techniques: Concentration Assay

    Acid secretory capacity declines in parallel with the decline in parietal cell number. Time course of acid secretory rates in isolated gastric mucosae of (a) 8‐day‐, (b) 1‐month‐ and (c) 6‐month‐old Car9 −/− and WT mice. (d) Basal and (e) maximal acid secretory rates after stimulation with forskolin ( FSK ) and IBMX were significantly lower in Car9 −/− mice than in WT up from 3 months of age. The decline in acid secretory capacity in the mucosa of Car9 −/− mice is paralleled by the decline in parietal cell numbers (see Fig. 2 ). n = 6–7, * P

    Journal: Acta Physiologica (Oxford, England)

    Article Title: Genetic ablation of carbonic anhydrase IX disrupts gastric barrier function via claudin‐18 downregulation and acid backflux

    doi: 10.1111/apha.12923

    Figure Lengend Snippet: Acid secretory capacity declines in parallel with the decline in parietal cell number. Time course of acid secretory rates in isolated gastric mucosae of (a) 8‐day‐, (b) 1‐month‐ and (c) 6‐month‐old Car9 −/− and WT mice. (d) Basal and (e) maximal acid secretory rates after stimulation with forskolin ( FSK ) and IBMX were significantly lower in Car9 −/− mice than in WT up from 3 months of age. The decline in acid secretory capacity in the mucosa of Car9 −/− mice is paralleled by the decline in parietal cell numbers (see Fig. 2 ). n = 6–7, * P

    Article Snippet: Maximal acid secretory capacity was investigated by serosal application of 10 μ m forskolin (FSK) and 100 μ m IBMX, both purchased from Alexis Biochemicals (Lörrach, Germany).

    Techniques: Isolation, Mouse Assay

    Expansion and trilineage differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). (A) Cell counts, (B) population doublings per day, and (C) cumulative population doublings at each passage during expansion of BMSCs under normoxia or hypoxia. Data points represent mean ± standard error of the mean of cells from six donors, and P values are listed. (D) Osteogenic differentiation of BMSCs verified with Alizarin Red S staining following expansion under hypoxia and monolayer culture within medium containing β-glycerophosphate, dexamethasone, and fetal bovine serum. (E) Adipogenic differentiation of BMSCs verified with Oil Red O staining following expansion under hypoxia and monolayer culture within medium containing isobutyl-1-methylxanthine (IBMX), indomethacin, dexamethasone, and fetal bovine serum. (F) Chondrogenic differentiation of BMSCs verified by safranin O staining following expansion under hypoxia and culture performed in pellets (left) or scaffolds composed of collagen (middle) or hyaluronic acid (right) submersed in a defined serum-free chondrogenic medium containing transforming growth factor-beta 3 and dexamethasone.

    Journal: Stem Cell Research & Therapy

    Article Title: Hypoxic culture of bone marrow-derived mesenchymal stromal stem cells differentially enhances in vitro chondrogenesis within cell-seeded collagen and hyaluronic acid porous scaffolds

    doi: 10.1186/s13287-015-0075-4

    Figure Lengend Snippet: Expansion and trilineage differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). (A) Cell counts, (B) population doublings per day, and (C) cumulative population doublings at each passage during expansion of BMSCs under normoxia or hypoxia. Data points represent mean ± standard error of the mean of cells from six donors, and P values are listed. (D) Osteogenic differentiation of BMSCs verified with Alizarin Red S staining following expansion under hypoxia and monolayer culture within medium containing β-glycerophosphate, dexamethasone, and fetal bovine serum. (E) Adipogenic differentiation of BMSCs verified with Oil Red O staining following expansion under hypoxia and monolayer culture within medium containing isobutyl-1-methylxanthine (IBMX), indomethacin, dexamethasone, and fetal bovine serum. (F) Chondrogenic differentiation of BMSCs verified by safranin O staining following expansion under hypoxia and culture performed in pellets (left) or scaffolds composed of collagen (middle) or hyaluronic acid (right) submersed in a defined serum-free chondrogenic medium containing transforming growth factor-beta 3 and dexamethasone.

    Article Snippet: Adipogenesis was then induced over the course of 3 days in 2.5 mL of basic culture medium supplemented with 1 μM dexamethasone, 0.5 mL insulin-transferrin-selenium (ITS) + 1, 100 μM indomethacin, and 500 μM isobutyl-1-methylxanthine (IBMX) (Sigma-Aldrich), followed by 1 day of culture in 2.5 mL of basic culture medium supplemented with only 0.5 mL ITS + 1.

    Techniques: Derivative Assay, Staining

    Comparison of ASC differentiation to adipocytes. Comparison of adipocyte differentiation between the WT ( Pin1 +/+ ; p53 -/- ) ASC and Pin1-KO ( Pin1 -/- ; p53 -/- ) ASC, and Pin1-KO ASC rescued with the lentiviral Pin1 cDNA. The wild type ASC, the Pin1-KO ASC- infected with Mock and the Pin1-KO ASC- infected with lentiviral Pin1 cDNA were cultured in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin for 0–16 days. ASCs were treated with 4% paraformaldehyde and 60% 2-propanol, and then stained with Oil Red O. The images of the oil red O-stained cells on 0, 4, 7, 10, 13, and 16 days after treatment with the differentiation reagent are shown.

    Journal: PLoS ONE

    Article Title: Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes

    doi: 10.1371/journal.pone.0168830

    Figure Lengend Snippet: Comparison of ASC differentiation to adipocytes. Comparison of adipocyte differentiation between the WT ( Pin1 +/+ ; p53 -/- ) ASC and Pin1-KO ( Pin1 -/- ; p53 -/- ) ASC, and Pin1-KO ASC rescued with the lentiviral Pin1 cDNA. The wild type ASC, the Pin1-KO ASC- infected with Mock and the Pin1-KO ASC- infected with lentiviral Pin1 cDNA were cultured in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin for 0–16 days. ASCs were treated with 4% paraformaldehyde and 60% 2-propanol, and then stained with Oil Red O. The images of the oil red O-stained cells on 0, 4, 7, 10, 13, and 16 days after treatment with the differentiation reagent are shown.

    Article Snippet: The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 0.5 mM 3-isobutyl-1-methylxanthine (Nacalai tesque), 1 μM dexamethasone (Sigma), and 1.7 μM insulin (Wako) with brown algae polyphenol (0–150 μg/ml) for a span of 16 days.

    Techniques: Infection, Cell Culture, Staining

    Effect of brown algae polyphenol on the differentiation of NIH3T3-L1 cells to adipocytes. A) The amount of the intracellular lipid stained with oil red O. NIH3T3-L1 cells were cultured in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin with 0 (diamond), 50 (square), 100 (triangle), and 150 μg/ml (x) brown algae polyphenol for 0–8 days. NIH3T3-L1 cells were treated with 4% paraformaldehyde and 60% 2-propanol and then stained with Oil Red O. The amount of oil red O extracted from the cells was determined three times by measuring absorbance at 550 nm (means ± SEM). B) PCR analysis of adipocyte biomarker mRNA levels. NIH3T3-L1 cells were cultured with 0, 50, 100, and 150 μg/ml of brown algae polyphenol for 0–8 days. The mRNA levels of PPARγ, C/EBPα, Glut4, FABP4, and LPL in these cells were compared by PCR.

    Journal: PLoS ONE

    Article Title: Brown Algae Polyphenol, a Prolyl Isomerase Pin1 Inhibitor, Prevents Obesity by Inhibiting the Differentiation of Stem Cells into Adipocytes

    doi: 10.1371/journal.pone.0168830

    Figure Lengend Snippet: Effect of brown algae polyphenol on the differentiation of NIH3T3-L1 cells to adipocytes. A) The amount of the intracellular lipid stained with oil red O. NIH3T3-L1 cells were cultured in DMEM containing 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1.7 μM insulin with 0 (diamond), 50 (square), 100 (triangle), and 150 μg/ml (x) brown algae polyphenol for 0–8 days. NIH3T3-L1 cells were treated with 4% paraformaldehyde and 60% 2-propanol and then stained with Oil Red O. The amount of oil red O extracted from the cells was determined three times by measuring absorbance at 550 nm (means ± SEM). B) PCR analysis of adipocyte biomarker mRNA levels. NIH3T3-L1 cells were cultured with 0, 50, 100, and 150 μg/ml of brown algae polyphenol for 0–8 days. The mRNA levels of PPARγ, C/EBPα, Glut4, FABP4, and LPL in these cells were compared by PCR.

    Article Snippet: The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 0.5 mM 3-isobutyl-1-methylxanthine (Nacalai tesque), 1 μM dexamethasone (Sigma), and 1.7 μM insulin (Wako) with brown algae polyphenol (0–150 μg/ml) for a span of 16 days.

    Techniques: Staining, Cell Culture, Polymerase Chain Reaction, Biomarker Assay

    Effects of late I Na induction on cellular cAMP levels in atrial CMs of mice. ( A–D ) YFP/CFP tracings in CMs of EPAC-camps transgenic mice upon ATX-II (0.5 nmol/L) and subsequent isoproterenol treatment (100 nmol/L). CMs were preincubated with a ( B ) PDE inhibitor IBMX (100 µmol/L), ( C ) an inhibitor of AC 5/6 (NKY80, 10 µmol/L), ( D ) an inhibitor of reverse-mode NCX KBR (0.1 µmol/L), or ( A ) no inhibitor. ( E ) Quantification of response of YFP/CFP ratio ( n mice/cells = 10/39 vs. 3/8 vs. 3/10 vs. 3/11). *significant vs. steady-state before ATX-II; # significant vs. YFP/CFP ratio upon ATX-II in the control group. * / # P

    Journal: Cardiovascular Research

    Article Title: Late INa increases diastolic SR-Ca2+-leak in atrial myocardium by activating PKA and CaMKII

    doi: 10.1093/cvr/cvv153

    Figure Lengend Snippet: Effects of late I Na induction on cellular cAMP levels in atrial CMs of mice. ( A–D ) YFP/CFP tracings in CMs of EPAC-camps transgenic mice upon ATX-II (0.5 nmol/L) and subsequent isoproterenol treatment (100 nmol/L). CMs were preincubated with a ( B ) PDE inhibitor IBMX (100 µmol/L), ( C ) an inhibitor of AC 5/6 (NKY80, 10 µmol/L), ( D ) an inhibitor of reverse-mode NCX KBR (0.1 µmol/L), or ( A ) no inhibitor. ( E ) Quantification of response of YFP/CFP ratio ( n mice/cells = 10/39 vs. 3/8 vs. 3/10 vs. 3/11). *significant vs. steady-state before ATX-II; # significant vs. YFP/CFP ratio upon ATX-II in the control group. * / # P

    Article Snippet: The cells were preincubated for 7 min with Tyrode solution containing IBMX (Applichem, 0.1 mmol/L), NKY80 (Sigma-Aldrich, 0.01 mmol/L), KBR (0.1 μmol/L), or no inhibitor in the control group.

    Techniques: Mouse Assay, Transgenic Assay

    Investigation of phosphodiesterase regulation of β 3 -AR/cGMP in adult cardiomyocytes. Representative FRET response curves of control (grey line) and failing (black line) cardiomyocytes following whole cell treatment with isoproterenol (100 nmol/L) followed by the PDE1 blocker vinpocetine (VINPO, 10 µmol/L) ( A ), the PDE2 inhibitor EHNA (10 µmol/L) ( B ), the PDE3 inhibitor cilostamide (CILO, 10 µmol/L) ( C ) and the PDE5 inhibitor tadalafil (TAD, 100 nmol /L) ( D ) followed by the non-specific PDE blocker IBMX (100 µmol/L). The scatter plot/histograms present whole cell cGMP-FRET responses evoked by PDE inhibition further to the isoproterenol responses in % from ( A–D ) ( E ) Error bars represent standard error of the mean. Numbers of cells/hearts are shown below the bars. Statistical significance was calculated via mixed ANOVA followed by χ2-test: No statistically significant differences between control and failing conditions for any PDE could be detected, only tendencies to increased responses for PDE2, PDE3 and PDE5 inhibitors. FRET microscopy data - 'whole cell' analysis PDEs.

    Journal: eLife

    Article Title: β3-Adrenoceptor redistribution impairs NO/cGMP/PDE2 signalling in failing cardiomyocytes

    doi: 10.7554/eLife.52221

    Figure Lengend Snippet: Investigation of phosphodiesterase regulation of β 3 -AR/cGMP in adult cardiomyocytes. Representative FRET response curves of control (grey line) and failing (black line) cardiomyocytes following whole cell treatment with isoproterenol (100 nmol/L) followed by the PDE1 blocker vinpocetine (VINPO, 10 µmol/L) ( A ), the PDE2 inhibitor EHNA (10 µmol/L) ( B ), the PDE3 inhibitor cilostamide (CILO, 10 µmol/L) ( C ) and the PDE5 inhibitor tadalafil (TAD, 100 nmol /L) ( D ) followed by the non-specific PDE blocker IBMX (100 µmol/L). The scatter plot/histograms present whole cell cGMP-FRET responses evoked by PDE inhibition further to the isoproterenol responses in % from ( A–D ) ( E ) Error bars represent standard error of the mean. Numbers of cells/hearts are shown below the bars. Statistical significance was calculated via mixed ANOVA followed by χ2-test: No statistically significant differences between control and failing conditions for any PDE could be detected, only tendencies to increased responses for PDE2, PDE3 and PDE5 inhibitors. FRET microscopy data - 'whole cell' analysis PDEs.

    Article Snippet: Experimental reagents M199 medium (Invitrogen UK, 11150), taurine (Biochemica, A1141), creatine monohydrate (Sigma Aldrich, C3630), penicillin/streptomycin (Merck, A2212), carnitine hydrochloride (Sigma Aldrich, C9500) BSA (Sigma Aldrich, A6003), laminin (Sigma Aldrich L2020), isoproterenol hydrochloride (Sigma Aldrich, I6504), ICI118551 (Tocris UK, 0821), CGP 20712A (Tocris UK, 1024), SR 59230A hydrochloride (Tocris UK, 1511), L-NAME hydrochloride (Tocris UK, 0665), Vinpocetine (Sigma Aldrich, V6383), EHNA hydrochloride (Sigma Aldrich, E114), Cilostamide (Tocris UK, 0915), Tadalafil (Santa Cruz USA, sc-208412), IBMX (Santa Cruz sc-201188), self-made rabbit sGCα and β subunit antibodies (specificity tested in KO animals; ), mouse α-actinin (Sigma Aldrich, A7732), mouse Caveolin-3 (BD Transduction Laboratories, 610421, specificity tested in KO animals; ), secondary Alexa Fluor antibodies 488 nm, 514 nm, 568 nm and 633 nm (Life Technologies), BSA (Fisher Scientific UK, BPE9704), fluorescence mounting medium (Vectashield Germany, H-1000), MaTek glass-bottom dishes (MaTek USA, P35G-1.5–10 C), TAT-scram and TAT-Cav3 peptides (a gift from Dr. Sarah Calaghan from Leeds, England).

    Techniques: Inhibition, Microscopy