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  • 99
    Millipore forskolin
    Pulmonary ionocytes are a major source of CFTR activity. a , Immunofluorescence for Krt5 (green) and Foxi1 (red) in mouse tracheae at homeostasis (left, n=3) or 3 days post injury (right, n=3). Arrowhead: Foxi1 + Krt5 + cells. Arrow: Foxi1 + Krt5 - cell. Scale bars, 20μm. b , Immunofluorescence and quantification for ionocytes (FOXI1 + , red, arrowheads) and ciliated cells (FOXJ1 + , green) in HBEC cultures treated with DMSO or DAPT, scale bar, 100 μm, (n=4 experiments in one donor). *p-value=0.01, ***p-value=1.1×10 −6 by two-tailed t-test c , HBECs were treated with DMSO or DAPT upon culture at ALI. After differentiation (2–3 weeks), cultures were loaded into Ussing chambers and short circuit current (I sc ) was recorded during addition of Amiloride, <t>Forskolin,</t> and a CFTR-inhibitor, CFTR(inh)-172. Shown is a representative tracing from Donor 1 (n=11). d , Change in short circuit current (ΔI sc ) in response to Forskolin measured in DMSO (n=7 cultures per donor) and DAPT-treated cultures, (n=8 cultures per donor). ***p-value
    Forskolin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 12375 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Tocris nkh 477
    G protein activation and βarr2 recruitment in absence and presence of active G proteins. a Inhibition of <t>forskolin-mediated</t> cAMP accumulation by C3-activated FFA2 in FFA2-HEK293 cells, in the presence or absence of PTX. b IP1 accumulation of C3-stimulated FFA2, in the presence or absence of FR. c , d FRET measurements of Gi ( c ) and Gq ( d ) activation-induced conformational changes in wild-type (WT) HEK293 cells expressing FFA2 after stimulation with C3 in absence and presence of G protein inhibitors. e , f C3-induced β-arrestin2 recruitment to FFA2 in wild-type (WT) ( e ) or ΔG12/13 HEK293 cells ( f ), in the absence and presence of PTX and FR. Shown are representative FRET traces with the indicated number of total cells per condition ( c , d ) and bar diagrams are averages from three independent experiments. Data are mean ± s.e.m. ( a , b , e , f ) or + s.d. ( c , d ) of three experiments each performed in triplicate. For statistical analysis, two-tailed unpaired t -test ( c , d ) and two-sample paired Wilcoxon test ( e , f ) was applied to paired points at different concentrations. * P
    Nkh 477, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical forskolin
    PAF-induced vasoconstriction. Intestines were stimulated with a bolus of 0.5 nmol PAF (n = 5). ( a ) Intracellular second messengers: AC stimulator <t>forskolin</t> plus the PDE inhibitor IBMX (Forsk/IBMX + PAF, n = 4),
    Forskolin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 333 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM forskolin
    Identification of CNN3 as a fusion-related protein. (A) BeWo cell fusion visualized by the conventional staining with anti–E-cadherin or anti-desmoplakin in combination with nuclear staining with DAPI. Cells were treated with or without 50 μM <t>forskolin</t> for 48 h. Arrows indicate a syncytium in which these marker proteins were translocated and diffused into the cytoplasm. Scale bar: 50 μm. (B) Detection of forskolin-induced syncytium between DsRed- and CFP-Nuc-expressing BeWo cells. A syncytium is easily recognized by the cyan-colored multiple nuclei within the homogeneously red-colored cytoplasm, whereas unfused cells have either red cytoplasm or cyan nuclei. Scale bar: 100 μm. (C) Heterologous fusion of CFP-Nuc–expressing HEK293 cells with BeWo cells expressing DsRed. The incidence of BeWo/HEK293 fusion was higher than that of homologous BeWo cell fusion, and the hybrid syncytium was much larger than that of BeWo-derived homologous syncytium. Scale bar: 250 μm. The broken lines in B and C show the periphery of the syncytium. (D) Cytoplasmically attached peripheral membrane proteins (CAPMPs) isolated from BeWo/HEK293 coculture were separated by 2-DE. The gels were stained with ProQ Diamond phospho-specific staining (top panels) or SYPRO Ruby protein staining (middle panels). A spot (arrow) among a series of protein spots in inset was subjected to in-gel digestion followed by peptide mass fingerprinting, and CNN3 was identified. All these spots in line were confirmed to be CNN3 by Western blotting (bottom panels). (E) Endogenous CNN3 was immunopurified using anti-CNN3 antibody from total cell lysate (TCL) of the cells incubated with 50 μM forskolin for the indicated times. The phosphorylation status of CNN3 was analyzed by ProQ Diamond phospho-specific staining, and the same gel was stained by Sypro Ruby to verify each sample equal in the amount of CNN3. “B + 293” indicates a BeWo/HEK293 mixed culture. (F) Western blot of CNN3 in the TCL or the CAPMP fraction from BeWo cells in the presence or absence of 50 μM forskolin for 72 h.
    Forskolin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 236 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem forskolin
    PKA C-α knockdown inhibits PGE 2 - and <t>forskolin-</t> induced IL-6 mRNA synthesis in human chondrocytes. T/C-28a2 chondrocytes were transfected with an small interfering RNA (siRNA) oligonucleotide sequence specific for PKA C-α before being treated with either PGE 2 (10 μM) or forskolin (20 μM) for 1 or 2 h. PKA C-α ( middle ) and IL-6 mRNA ( bottom ) expressions were determined by quantitative RT-PCR. GAPDH was served as internal control. Data are means ± SE of three independent experiments. * P
    Forskolin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    LC Laboratories forskolin
    Pharmacologic ATR inhibition does not interfere with cAMP-induced Mitf activation SK-MEL-2 melanoma cells (n=3) (A–B) and PHMs (n=3) (C–D) were treated with 10 uM <t>forskolin,</t> VE821, or a combination of 10 uM forskolin and 10 uM VE-821. Whole cell lysates were collected 1 hour following treatment and immunoblotted for Mitf. B,D) Composite densitometry quantification of immunoblots (n=3 per condition) in A,C. NS denotes that no significant difference was observed between experimental groups as determined by one-way ANOVA and Tukey post hoc test. Data are expressed as mean-fold change over control ± SEM.
    Forskolin, supplied by LC Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Abcam forskolin
    Target dependent effects of miR-449a on proliferation, differentiation and glioma biology ( a ) in stemness—maintaining growth conditions with EGF, FGF enriched serum-free medium, miR-449a—mediated effects are predominantly exerted through CCND1 inhibition, thus reducing invasion and proliferation. For example, miR-449a high Rb/p53 cells have low expression levels of Ccdn1, proliferate and migrate slower, and have less self-renewal capacity. b in growth conditions promoting neural phenotypes in vitro, such as FBS, <t>Forskolin,</t> retinoic acid and in vivo experimental settings and humans gliomas, miR-449a suppresses GPR158, reducing neural marker expression, and is associated with higher glioma grade and shorter survival. Numbers and letters provide a reference to figures in the text
    Forskolin, supplied by Abcam, used in various techniques. Bioz Stars score: 94/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Cell Signaling Technology Inc forskolin
    Immunoblot analysis of protein kinase A (PKA) substrates in T cells. (A) HuT78 cells were serum starved for 4 h and then treated with DMSO ( control ) or 30 µM <t>forskolin</t> for 30 min and lysed. Protein lysates were immunoprecipitated (IP) using either phospho-PKA substrate antibody ( p-PKA substrate ). (B) Control and CG-NAP knockdown ( KD ) HuT78 T-cells were either unstimulated or LFA-1-stimulated (+rICAM-1) for 1 h, lysed and IP using anti-PKARIIα antibody. Immunoprecipitates were resolved on SDS-PAGE and subjected to Western blotting with anti-CG-NAP, pericentrin, dynein, or PKARIIα antibodies. (C) Control or CG-NAP KD HuT78 cells were treated with 30 µM forskolin for 30 min and protein lysates were IP using either phospho-PKA substrate antibody or control IgG. Immunoprecipitates were resolved on SDS-PAGE and subjected to Western blotting with anti-pericentrin and anti-dynein antibodies. Whole cell lysates ( WCL , 10 µg each) were used as input controls for Western immunoblots; gel lanes indicated by “X” are empty lanes, i.e., no protein loaded. Relative densitometry graph of Western immunoblots obtained from at least three independent experiments are presented (mean ± SEM, * p
    Forskolin, supplied by Cell Signaling Technology Inc, 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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    92
    Biomol GmbH forskolin
    Glucocorticoid receptor (GR) expression in CEM and MM.1 cells. (A,B,E) Quantitative real-time PCR analysis of GR mRNA expression in CEM-S2 (A) , CEM-R8 (B) , and MM.1S (E) cells, following treatment with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM <t>forskolin</t> (R+F), or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+D) at different times as indicated. Data represent the mean ± SD of at least two independent experiments assayed in triplicate, shown as fold change relative to control. (C,D,F) Western blot analysis of GR protein expression in CEM-S2 (C) , CEM-R8 (D) , and MM.1S (F) cells following treatment at different times with dexamethasone and/or rolipram and forskolin as indicated. Equal amounts of protein (20 μg) from whole cell lysates were added per lane. Data shown represents one of at least two independent experiments with similar results. The numbers at the top of the Western blot images represent the GR band densities following different treatments relative to control, normalized based on the density of the GAPDH housekeeping protein, calculated as described in Section “Materials and Methods.”
    Forskolin, supplied by Biomol GmbH, used in various techniques. Bioz Stars score: 92/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Selleck Chemicals forskolin
    Corrector treatment increases CFTR activation response of nonsense variants in exon 22 that result in mature truncated CFTR. (A) Schematic of CFTR-Expression Minigene with abridged introns 21 and abridged intron 22 (EMG-i21-i22) constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and two abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (B) RT-qPCR showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Steady state levels of CFTR protein from HEK293 cells transiently transfected with wild-type EMG or EMGs with different nonsense variants. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody-MM13-4 (EMD Millipore). (D) Representative IB showing sensitivity of CFTR to PNGaseF and Endo H. Mature complex glycosylated band is sensitive to PNGase only, whereas immature core glycosylated band is sensitive to both PNGase and EndoH. (E). Schematic illustration of the nonsense variants in the protein context showing their classification into two groups based on mRNA stability and protein maturity. Each nonsense variant truncates CFTR at intracellular loop 6 (ICL6) just before NBD2. (F) A representative Ussing chamber tracing of CFBE cells stably expressing S1196X-EMG. Short-circuit (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. ( G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with <t>forskolin</t> alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated I sc without modulator treatment in cells expressing EMG i21-i22. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001), n.s. (not significant, P > 0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.
    Forskolin, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 99/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris nkh477
    Spatio-temporal cAMP dynamics in the sperm flagellum. ( A ) cAMP dynamics was analyzed in a region 20 µm in length in the midpiece (blue) and principal piece (green) of freely beating sperm. The cytoplasmic droplet is indicated with an arrow. ( B ) Changes in FRET after stimulation with 40 μM <t>NKH477.</t> The perfusion with NKH477 is indicated with a grey box. Data for a representative cell are shown as mean ± S.D. in the midpiece (blue) and principal piece (green). ( C ) Changes in FRET after stimulation with 25 mM bicarbonate. The perfusion with bicarbonate is indicated with a blue box. Individual traces have been fitted using logistic regression (Origin 9) (red line). ( D ) Average of the fitted data presented in ( C ). The blue and green line represent the mean value for the midpiece and principal piece, respectively (n = 7). The blue and green areas represent the corresponding S.D. DOI: http://dx.doi.org/10.7554/eLife.14052.013
    Nkh477, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    thermo fisher forskolin
    Rapid generation of human GABAergic neurons by overexpression of Ascl1 and <t>forskolin.</t> ( a ) Culturing paradigm for the generation of induced GABAergic neurons (iGABAA-FSK). ( b ) iGABA A-FSK neuron immunostaining at DIV 49 for neuronal marker MAP2 co-labelled with Glutamate decarboxylase (GAD) 67 or GABA. ( c ) iGABA A-FSK are co-cultured from DIV 0 on with iGLU Ngn2 to promote functional maturation (named E/I networks), in a ratio of E/I 65:35. ( d ) VGAT and Gephyrin co-localisation in E/I networks at DIV 49. ( e ) Immunostaining for GABA co-labelled with either calbindin (CB), calretinin (CR), somatostatin (SST), parvalbumin (PV), MEF2C (asterix) or synaptotagmin-2 (SYT2, arrowheads) in E/I networks (Quantification sample size n= 7-9 coverslips per condition). ( f ) Heatmap showing expression of GABAergic/Glutamatergic transporters and – subtypes genes, and expression of genes important in GABAergic neuron development in E/I 65:35 networks at DIV 49 (3 biological replicates from one neuronal preparation). Data represents the log-transformed counts per million (logCPM). ( g ) Representative firing patterns of iGABA A+FSK neurons at DIV 28, −35 and −49. ( h-i ) Analysis of iGABA A+FSK membrane properties including ( h ) resting membrane potential (Vrmp) and ( i ) membrane capacitance (Cm). ( j-k ) Analysis of action potentials evoked by step-depolarization of iGABA A-FSK membranes including ( j ) fractions of maximum number of action potentials, and ( k ) Rheobase. ( l ) Quantifications of correlated synaptic input (number of synaptic burst/minute). ( m ) Spontaneous glutamatergic (red inset) and GABAergic (blue inset) postsynaptic inputs (sPSCs) received by iGLU Ngn2 . ( n ) Quantification of synaptic input types (Sample size for DIV 28 n=39, DIV 35 n=38, DIV 49 n=41 cells from 3 batches). All data represent means ± SEM. * p
    Forskolin, supplied by thermo fisher, used in various techniques. Bioz Stars score: 93/100, based on 521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck KGaA forskolin
    Blockade of ramelteon-induced potentiation of CREB phosphorylation by luzindole and <t>forskolin.</t> (A) INS-1 cells were incubated with ramelteon (5 nM) in the absence or presence of luzindole (15 µM) or forskolin (0.1 µM) for 14 h. After drug washout, the cells were subjected to a second round of forskolin stimulation (0.1 µM) for 30 min. (B) INS-1 cells were incubated with 2′,5′-dideoxyadenosine (10 or 100 µM) and H89 (1 or 10 µM) for 2 or 14 h. After drug washout, the cells were stimulated with forskolin (0.1 µM) for 30 min. Values are expressed as the ratio of phosphorylated CREB to total CREB in the vehicle-pretreated control (100%). Data are presented as means ± SEM (n = 3–6) and were analyzed using 2-way analysis of variance followed by Dunnett's test. *** P
    Forskolin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 173 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology forskolin
    PKA acts downstream R-Smads to mediate anti-motility signaling of TGFβ3. ( A ) HDFs were infected with FG-12 lentivirus carrying either a GFP control gene or a shRNAs against PKA-Cα. After 48 hours, downregulation of the endogenous PKA-Cα was confirmed by immunoblotting the lysates of the cells with an anti-PKA-Cα antibody. ( B ) Downregulation of PKA-Cα blocks <t>forskolin-stimulated</t> CREB phosphorylation (lane 4 versus lane 2). ( C ) Downregulation of PKA-Cα showed little effect on TGFβ3-stimulated phosphorylation of Smad3 (lanes 4 and 6 versus lane 2). ( D ) Two sets of PKA-Cα-downregulated HDFs were subjected to colloidal gold migration assay in response to PDGF-bb (15 ng/ml) in the absence or presence of TGFβ3 (3 ng/ml). Representative images of the migrated cells under the indicated conditions are shown (a to i). ( E ) The computer-assisted quantitative analyses of the migration tracks are shown as Migration index (MI) (%). The experiment was repeated four times ( n = 3, P ≤0.05). *Statistically significant over the control.
    Forskolin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific forskolin
    I SC and C T recordings with and without hormonal supplementation. (A) Representative I SC (black trace) and C T (grey trace) recording from mpkCCD cells mounted in modified Ussing chambers and stimulated with (10 µM) <t>forskolin.</t> Addition of 10 µM amiloride at the end of the trace demonstrated the majority of the recorded I SC was Na + transport via ENaC. (B) A similar trace from mpkCCD cells cultured in the absence of dexamethasone supplementation. (C) Summarized data for stimulated amiloride-sensitive current (I Na ) and C T response to forskolin stimulation (n = 14) in cells with (+) and without (-) full supplementation.
    Forskolin, supplied by Fisher Scientific, 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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    92
    Merck & Co forskolin
    Involvement of PKC isoforms in EPAC-dependent SOCS-3 induction in COS1 cells. a , COS1 cells were stimulated for 5 h with MG132 (10 μ m ) plus 50 μ m 8Me, a combination of 10 μ m <t>forskolin</t> plus 10 μ m rolipram (F/R) or 10 μ
    Forskolin, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Pulmonary ionocytes are a major source of CFTR activity. a , Immunofluorescence for Krt5 (green) and Foxi1 (red) in mouse tracheae at homeostasis (left, n=3) or 3 days post injury (right, n=3). Arrowhead: Foxi1 + Krt5 + cells. Arrow: Foxi1 + Krt5 - cell. Scale bars, 20μm. b , Immunofluorescence and quantification for ionocytes (FOXI1 + , red, arrowheads) and ciliated cells (FOXJ1 + , green) in HBEC cultures treated with DMSO or DAPT, scale bar, 100 μm, (n=4 experiments in one donor). *p-value=0.01, ***p-value=1.1×10 −6 by two-tailed t-test c , HBECs were treated with DMSO or DAPT upon culture at ALI. After differentiation (2–3 weeks), cultures were loaded into Ussing chambers and short circuit current (I sc ) was recorded during addition of Amiloride, Forskolin, and a CFTR-inhibitor, CFTR(inh)-172. Shown is a representative tracing from Donor 1 (n=11). d , Change in short circuit current (ΔI sc ) in response to Forskolin measured in DMSO (n=7 cultures per donor) and DAPT-treated cultures, (n=8 cultures per donor). ***p-value

    Journal: Nature

    Article Title: A single cell atlas of the tracheal epithelium reveals the CFTR-rich pulmonary ionocyte

    doi: 10.1038/s41586-018-0394-6

    Figure Lengend Snippet: Pulmonary ionocytes are a major source of CFTR activity. a , Immunofluorescence for Krt5 (green) and Foxi1 (red) in mouse tracheae at homeostasis (left, n=3) or 3 days post injury (right, n=3). Arrowhead: Foxi1 + Krt5 + cells. Arrow: Foxi1 + Krt5 - cell. Scale bars, 20μm. b , Immunofluorescence and quantification for ionocytes (FOXI1 + , red, arrowheads) and ciliated cells (FOXJ1 + , green) in HBEC cultures treated with DMSO or DAPT, scale bar, 100 μm, (n=4 experiments in one donor). *p-value=0.01, ***p-value=1.1×10 −6 by two-tailed t-test c , HBECs were treated with DMSO or DAPT upon culture at ALI. After differentiation (2–3 weeks), cultures were loaded into Ussing chambers and short circuit current (I sc ) was recorded during addition of Amiloride, Forskolin, and a CFTR-inhibitor, CFTR(inh)-172. Shown is a representative tracing from Donor 1 (n=11). d , Change in short circuit current (ΔI sc ) in response to Forskolin measured in DMSO (n=7 cultures per donor) and DAPT-treated cultures, (n=8 cultures per donor). ***p-value

    Article Snippet: Amiloride (Sigma, A9561) was added apically at 10μM to inhibit Na+ absorption, then Forskolin (Sigma, F6886) was added apically at 20μM to stimulate cAMP and finally, CFTR-172 (Sigma-Aldrich, C2992) inhibitor was added apically and basally at 30μM.

    Techniques: Activity Assay, Immunofluorescence, Two Tailed Test

    G protein activation and βarr2 recruitment in absence and presence of active G proteins. a Inhibition of forskolin-mediated cAMP accumulation by C3-activated FFA2 in FFA2-HEK293 cells, in the presence or absence of PTX. b IP1 accumulation of C3-stimulated FFA2, in the presence or absence of FR. c , d FRET measurements of Gi ( c ) and Gq ( d ) activation-induced conformational changes in wild-type (WT) HEK293 cells expressing FFA2 after stimulation with C3 in absence and presence of G protein inhibitors. e , f C3-induced β-arrestin2 recruitment to FFA2 in wild-type (WT) ( e ) or ΔG12/13 HEK293 cells ( f ), in the absence and presence of PTX and FR. Shown are representative FRET traces with the indicated number of total cells per condition ( c , d ) and bar diagrams are averages from three independent experiments. Data are mean ± s.e.m. ( a , b , e , f ) or + s.d. ( c , d ) of three experiments each performed in triplicate. For statistical analysis, two-tailed unpaired t -test ( c , d ) and two-sample paired Wilcoxon test ( e , f ) was applied to paired points at different concentrations. * P

    Journal: Nature Communications

    Article Title: Lack of beta-arrestin signaling in the absence of active G proteins

    doi: 10.1038/s41467-017-02661-3

    Figure Lengend Snippet: G protein activation and βarr2 recruitment in absence and presence of active G proteins. a Inhibition of forskolin-mediated cAMP accumulation by C3-activated FFA2 in FFA2-HEK293 cells, in the presence or absence of PTX. b IP1 accumulation of C3-stimulated FFA2, in the presence or absence of FR. c , d FRET measurements of Gi ( c ) and Gq ( d ) activation-induced conformational changes in wild-type (WT) HEK293 cells expressing FFA2 after stimulation with C3 in absence and presence of G protein inhibitors. e , f C3-induced β-arrestin2 recruitment to FFA2 in wild-type (WT) ( e ) or ΔG12/13 HEK293 cells ( f ), in the absence and presence of PTX and FR. Shown are representative FRET traces with the indicated number of total cells per condition ( c , d ) and bar diagrams are averages from three independent experiments. Data are mean ± s.e.m. ( a , b , e , f ) or + s.d. ( c , d ) of three experiments each performed in triplicate. For statistical analysis, two-tailed unpaired t -test ( c , d ) and two-sample paired Wilcoxon test ( e , f ) was applied to paired points at different concentrations. * P

    Article Snippet: The cells were treated with vehicle, 100 nM arginine vasopressin (Peptide Institutes, Japan) or 100 µM NKH-477 (Tocris) (10 µL of 5X solution per well).

    Techniques: Activation Assay, Inhibition, Expressing, Two Tailed Test

    DMR biosensing of G protein- and arrestin-mediated cellular responses. DMR recordings in absence ( a – c ) and presence of G protein inhibitors ( d – f ) of agonist-mediated DP2 ( a , d ), GPR17 ( b , e ) and FFA2 ( c , f ) in wild-type (WT) HEK293 cells. Concentration-response-curves of DMR peak values in absence and presence of G protein inhibitors of DP2 ( g ), GPR17 ( h ), and FFA2 ( i ) response in wild-type HEK293 cells. j C3-mediated and forskolin-mediated DMR response of FFA2 in ΔG12/13 cells in the absence and presence of PTX/FR. k C3-induced DMR response of FFA2 in vector- or Gα 12/13 -transfected ΔG12/13 cells. l C3- or forskolin-mediated DMR response of FFA2 in vector- or β-arrestin2-transfected ΔG12/13 cells in the presence of PTX/FR. Inset shows βarr2-GFP transfected ΔG12/13 cells, scale bar 50 µm. m – o Agonist-induced DMR response of DP2 ( m ), GPR17 ( n ), or FFA2 ( o ) in vector- or β-arrestin2-transfected Δβarr1/2 cells in absence and presence of G protein inhibitors. a – f , j – o Shown are representative traces (mean + s.e.m.) of three independent experiments, each performed in triplicate. g – i Data are mean ± s.e.m. of three independent experiments (three technical replicates)

    Journal: Nature Communications

    Article Title: Lack of beta-arrestin signaling in the absence of active G proteins

    doi: 10.1038/s41467-017-02661-3

    Figure Lengend Snippet: DMR biosensing of G protein- and arrestin-mediated cellular responses. DMR recordings in absence ( a – c ) and presence of G protein inhibitors ( d – f ) of agonist-mediated DP2 ( a , d ), GPR17 ( b , e ) and FFA2 ( c , f ) in wild-type (WT) HEK293 cells. Concentration-response-curves of DMR peak values in absence and presence of G protein inhibitors of DP2 ( g ), GPR17 ( h ), and FFA2 ( i ) response in wild-type HEK293 cells. j C3-mediated and forskolin-mediated DMR response of FFA2 in ΔG12/13 cells in the absence and presence of PTX/FR. k C3-induced DMR response of FFA2 in vector- or Gα 12/13 -transfected ΔG12/13 cells. l C3- or forskolin-mediated DMR response of FFA2 in vector- or β-arrestin2-transfected ΔG12/13 cells in the presence of PTX/FR. Inset shows βarr2-GFP transfected ΔG12/13 cells, scale bar 50 µm. m – o Agonist-induced DMR response of DP2 ( m ), GPR17 ( n ), or FFA2 ( o ) in vector- or β-arrestin2-transfected Δβarr1/2 cells in absence and presence of G protein inhibitors. a – f , j – o Shown are representative traces (mean + s.e.m.) of three independent experiments, each performed in triplicate. g – i Data are mean ± s.e.m. of three independent experiments (three technical replicates)

    Article Snippet: The cells were treated with vehicle, 100 nM arginine vasopressin (Peptide Institutes, Japan) or 100 µM NKH-477 (Tocris) (10 µL of 5X solution per well).

    Techniques: Concentration Assay, Plasmid Preparation, Transfection

    Antibodies binding the glycine-rich loop of C-subunits present a cAMP-dependent intensity pattern in sensory neurons. (A) Time course of Cα intensity after stimulation with Ctrl (0.1% DMSO), 10 µM Fsk, 10 µM Sp-8-Br-cAMPS-AM, or 200 nM 5-HT. (B) ). (C) Cell density plots showing single-cell data of pRII/Cα–labeled neurons stimulated for 1, 5, or 30 min with 10 µM Fsk, 10 µM Sp-8-Br-cAMPS-AM, or 0.2 µM 5-HT. The Spearman’s rank correlation coefficient is shown. (D) Controls indicating that spillover between fluorescence channels was properly compensated to avoid false correlations. HCS results in A show means ± SEM; n = 3–4; > 1,000 neurons/condition; two-way ANOVA with Bonferroni’s test. ***, P

    Journal: The Journal of Cell Biology

    Article Title: PKA-RII subunit phosphorylation precedes activation by cAMP and regulates activity termination

    doi: 10.1083/jcb.201708053

    Figure Lengend Snippet: Antibodies binding the glycine-rich loop of C-subunits present a cAMP-dependent intensity pattern in sensory neurons. (A) Time course of Cα intensity after stimulation with Ctrl (0.1% DMSO), 10 µM Fsk, 10 µM Sp-8-Br-cAMPS-AM, or 200 nM 5-HT. (B) ). (C) Cell density plots showing single-cell data of pRII/Cα–labeled neurons stimulated for 1, 5, or 30 min with 10 µM Fsk, 10 µM Sp-8-Br-cAMPS-AM, or 0.2 µM 5-HT. The Spearman’s rank correlation coefficient is shown. (D) Controls indicating that spillover between fluorescence channels was properly compensated to avoid false correlations. HCS results in A show means ± SEM; n = 3–4; > 1,000 neurons/condition; two-way ANOVA with Bonferroni’s test. ***, P

    Article Snippet: 10 mM fentanyl (in dH2 O) and 10 mM Fsk (in DMSO) were from Tocris. cAMP, PO4 -AM3 , Sp-8-Br-cAMPS, Sp-8-Br-cAMPS-AM, Rp-cAMPS, Rp-8-Br-cAMPS, and Rp-8-pCPT-cAMPS (all 10 mM in DMSO) were from BioLog.

    Techniques: Binding Assay, Labeling, Fluorescence

    PAF-induced vasoconstriction. Intestines were stimulated with a bolus of 0.5 nmol PAF (n = 5). ( a ) Intracellular second messengers: AC stimulator forskolin plus the PDE inhibitor IBMX (Forsk/IBMX + PAF, n = 4),

    Journal: Scientific Reports

    Article Title: Signalling mechanisms in PAF-induced intestinal failure

    doi: 10.1038/s41598-017-13850-x

    Figure Lengend Snippet: PAF-induced vasoconstriction. Intestines were stimulated with a bolus of 0.5 nmol PAF (n = 5). ( a ) Intracellular second messengers: AC stimulator forskolin plus the PDE inhibitor IBMX (Forsk/IBMX + PAF, n = 4),

    Article Snippet: Forskolin (CAS: 66575-29-9) was obtained from Cayman Chemical Company (Ann Arbor, United States) and Y-27632 (CAS: 146986-50-7) was obtained from Tocris Bioscience (Bristol, United Kingdom).

    Techniques:

    Identification of CNN3 as a fusion-related protein. (A) BeWo cell fusion visualized by the conventional staining with anti–E-cadherin or anti-desmoplakin in combination with nuclear staining with DAPI. Cells were treated with or without 50 μM forskolin for 48 h. Arrows indicate a syncytium in which these marker proteins were translocated and diffused into the cytoplasm. Scale bar: 50 μm. (B) Detection of forskolin-induced syncytium between DsRed- and CFP-Nuc-expressing BeWo cells. A syncytium is easily recognized by the cyan-colored multiple nuclei within the homogeneously red-colored cytoplasm, whereas unfused cells have either red cytoplasm or cyan nuclei. Scale bar: 100 μm. (C) Heterologous fusion of CFP-Nuc–expressing HEK293 cells with BeWo cells expressing DsRed. The incidence of BeWo/HEK293 fusion was higher than that of homologous BeWo cell fusion, and the hybrid syncytium was much larger than that of BeWo-derived homologous syncytium. Scale bar: 250 μm. The broken lines in B and C show the periphery of the syncytium. (D) Cytoplasmically attached peripheral membrane proteins (CAPMPs) isolated from BeWo/HEK293 coculture were separated by 2-DE. The gels were stained with ProQ Diamond phospho-specific staining (top panels) or SYPRO Ruby protein staining (middle panels). A spot (arrow) among a series of protein spots in inset was subjected to in-gel digestion followed by peptide mass fingerprinting, and CNN3 was identified. All these spots in line were confirmed to be CNN3 by Western blotting (bottom panels). (E) Endogenous CNN3 was immunopurified using anti-CNN3 antibody from total cell lysate (TCL) of the cells incubated with 50 μM forskolin for the indicated times. The phosphorylation status of CNN3 was analyzed by ProQ Diamond phospho-specific staining, and the same gel was stained by Sypro Ruby to verify each sample equal in the amount of CNN3. “B + 293” indicates a BeWo/HEK293 mixed culture. (F) Western blot of CNN3 in the TCL or the CAPMP fraction from BeWo cells in the presence or absence of 50 μM forskolin for 72 h.

    Journal: Molecular Biology of the Cell

    Article Title: Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

    doi: 10.1091/mbc.E10-03-0261

    Figure Lengend Snippet: Identification of CNN3 as a fusion-related protein. (A) BeWo cell fusion visualized by the conventional staining with anti–E-cadherin or anti-desmoplakin in combination with nuclear staining with DAPI. Cells were treated with or without 50 μM forskolin for 48 h. Arrows indicate a syncytium in which these marker proteins were translocated and diffused into the cytoplasm. Scale bar: 50 μm. (B) Detection of forskolin-induced syncytium between DsRed- and CFP-Nuc-expressing BeWo cells. A syncytium is easily recognized by the cyan-colored multiple nuclei within the homogeneously red-colored cytoplasm, whereas unfused cells have either red cytoplasm or cyan nuclei. Scale bar: 100 μm. (C) Heterologous fusion of CFP-Nuc–expressing HEK293 cells with BeWo cells expressing DsRed. The incidence of BeWo/HEK293 fusion was higher than that of homologous BeWo cell fusion, and the hybrid syncytium was much larger than that of BeWo-derived homologous syncytium. Scale bar: 250 μm. The broken lines in B and C show the periphery of the syncytium. (D) Cytoplasmically attached peripheral membrane proteins (CAPMPs) isolated from BeWo/HEK293 coculture were separated by 2-DE. The gels were stained with ProQ Diamond phospho-specific staining (top panels) or SYPRO Ruby protein staining (middle panels). A spot (arrow) among a series of protein spots in inset was subjected to in-gel digestion followed by peptide mass fingerprinting, and CNN3 was identified. All these spots in line were confirmed to be CNN3 by Western blotting (bottom panels). (E) Endogenous CNN3 was immunopurified using anti-CNN3 antibody from total cell lysate (TCL) of the cells incubated with 50 μM forskolin for the indicated times. The phosphorylation status of CNN3 was analyzed by ProQ Diamond phospho-specific staining, and the same gel was stained by Sypro Ruby to verify each sample equal in the amount of CNN3. “B + 293” indicates a BeWo/HEK293 mixed culture. (F) Western blot of CNN3 in the TCL or the CAPMP fraction from BeWo cells in the presence or absence of 50 μM forskolin for 72 h.

    Article Snippet: Differentiation was induced by treatment with 50 μM forskolin (Wako), for up to 96 h ( ; ; ) with daily exchange of the forskolin-containing medium.

    Techniques: Staining, Marker, Expressing, Derivative Assay, Isolation, Peptide Mass Fingerprinting, Western Blot, Incubation

    CNN3 phosphorylation in BeWo cell fusion. (A) Comparison of the amino acid sequences of human CNN family proteins. CNN1 phosphorylation sites are indicated by asterisks. The sequence of ΔC mutant is truncated after Cys274 (arrow). (B) HEK293 cells transiently expressing WT or ΔC CNN3. Flag-CNN3 with a wild or Δ C sequence was recovered by immunoprecipitation from cell lysate using anti-Flag-agarose beads. The eluate was subjected to SDS-PAGE, and the phosphorylated CNN3s were visualized by ProQ Diamond phospho-specific staining (upper panel) followed by Sypro Ruby protein staining (lower panel). Note that the ΔC mutant was not phosphorylated but retained the actin-binding property. (C) Flag-tagged WT or ΔC CNN3 identified by Western blotting using anti-FLAG (left panel) or anti-CNN3 (right panel) antibodies. Each 20-μg sample of HEK293 cell lysate was loaded. (D and E) Phosphorylation levels of WT or mutant Flag-CNN3 transiently expressed in HEK293 cells. Flag-CNN3 was immunopurified using anti–Flag-agarose beads and was subjected to SDS-PAGE followed by ProQ Diamond phospho-specific staining. The ratio of phosphorylated and total CNN3 was calculated and indicated below each lane, showing decreased phosphorylation of S293A and S296A CNN3s. (F and G) BeWo cell fusion at 96 h after forskolin treatment in various mutants. The fusion frequency was counted as “fusion induction” (F), and the degree of maturation was calculated as “fusion index” (G). Data from three independent experiments were averaged, and standard deviations are indicated by error bars.

    Journal: Molecular Biology of the Cell

    Article Title: Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

    doi: 10.1091/mbc.E10-03-0261

    Figure Lengend Snippet: CNN3 phosphorylation in BeWo cell fusion. (A) Comparison of the amino acid sequences of human CNN family proteins. CNN1 phosphorylation sites are indicated by asterisks. The sequence of ΔC mutant is truncated after Cys274 (arrow). (B) HEK293 cells transiently expressing WT or ΔC CNN3. Flag-CNN3 with a wild or Δ C sequence was recovered by immunoprecipitation from cell lysate using anti-Flag-agarose beads. The eluate was subjected to SDS-PAGE, and the phosphorylated CNN3s were visualized by ProQ Diamond phospho-specific staining (upper panel) followed by Sypro Ruby protein staining (lower panel). Note that the ΔC mutant was not phosphorylated but retained the actin-binding property. (C) Flag-tagged WT or ΔC CNN3 identified by Western blotting using anti-FLAG (left panel) or anti-CNN3 (right panel) antibodies. Each 20-μg sample of HEK293 cell lysate was loaded. (D and E) Phosphorylation levels of WT or mutant Flag-CNN3 transiently expressed in HEK293 cells. Flag-CNN3 was immunopurified using anti–Flag-agarose beads and was subjected to SDS-PAGE followed by ProQ Diamond phospho-specific staining. The ratio of phosphorylated and total CNN3 was calculated and indicated below each lane, showing decreased phosphorylation of S293A and S296A CNN3s. (F and G) BeWo cell fusion at 96 h after forskolin treatment in various mutants. The fusion frequency was counted as “fusion induction” (F), and the degree of maturation was calculated as “fusion index” (G). Data from three independent experiments were averaged, and standard deviations are indicated by error bars.

    Article Snippet: Differentiation was induced by treatment with 50 μM forskolin (Wako), for up to 96 h ( ; ; ) with daily exchange of the forskolin-containing medium.

    Techniques: Sequencing, Mutagenesis, Expressing, Immunoprecipitation, SDS Page, Staining, Binding Assay, Western Blot

    Phosphorylation status and subcellular distribution of endogenous CNN3. (A) Site-specific phosphorylation of CNN3. Each 20-μg sample of the cytosolic or plasma membrane (PM) fraction from BeWo cells was subjected to SDS-PAGE followed by Western blotting using antibodies against specific phosphorylation site at S293 or S296 (see Supplemental Fig. S3 for their specificity). Enolase (cytosol) and E-cadherin (PM) were used as the control for cell fractionation. (B) Coimmunoprecipitation of actin with CNN3. Endogenous CNN3 was immunopurified from BeWo and/or HEK293 cells after forskolin treatment. Immuno-purified CNN3 or total cell lysates (TCL) were analyzed by Western blotting for actin or CNN3. (C) Dissociation of endogenous CNN3 from actin cytoskeleton in BeWo cell fusion. CNN3 was immunostained using rabbit anti-CNN3 IgG, and the cells were visualized by Alexa Fluor 488-conjugated secondary antibody (green). Subsequently, Alexa Fluor 568-conjugated phalloidin (red) and DAPI staining was performed (blue). Endogenous CNN3 in multinucleated BeWo cells was dissociated from actin cytoskeleton after forskolin treatment. Broken line shows the periphery of syncytium. Scale bar: 50 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

    doi: 10.1091/mbc.E10-03-0261

    Figure Lengend Snippet: Phosphorylation status and subcellular distribution of endogenous CNN3. (A) Site-specific phosphorylation of CNN3. Each 20-μg sample of the cytosolic or plasma membrane (PM) fraction from BeWo cells was subjected to SDS-PAGE followed by Western blotting using antibodies against specific phosphorylation site at S293 or S296 (see Supplemental Fig. S3 for their specificity). Enolase (cytosol) and E-cadherin (PM) were used as the control for cell fractionation. (B) Coimmunoprecipitation of actin with CNN3. Endogenous CNN3 was immunopurified from BeWo and/or HEK293 cells after forskolin treatment. Immuno-purified CNN3 or total cell lysates (TCL) were analyzed by Western blotting for actin or CNN3. (C) Dissociation of endogenous CNN3 from actin cytoskeleton in BeWo cell fusion. CNN3 was immunostained using rabbit anti-CNN3 IgG, and the cells were visualized by Alexa Fluor 488-conjugated secondary antibody (green). Subsequently, Alexa Fluor 568-conjugated phalloidin (red) and DAPI staining was performed (blue). Endogenous CNN3 in multinucleated BeWo cells was dissociated from actin cytoskeleton after forskolin treatment. Broken line shows the periphery of syncytium. Scale bar: 50 μm.

    Article Snippet: Differentiation was induced by treatment with 50 μM forskolin (Wako), for up to 96 h ( ; ; ) with daily exchange of the forskolin-containing medium.

    Techniques: SDS Page, Western Blot, Cell Fractionation, Purification, Staining

    CNN3 gene knockdown. (A) Control or CNN3-specific shRNA was introduced into BeWo cells expressing EYFP or CFP-Nuc. After washing, different marker expressing cells were trypsinized and mixed in the culture in the absence or presence of 50 μM forskolin for 72 h. After fixation, cells were stained with Alexa Fluor 568-conjugated phalloidin (red). Scale bar: 40 μm. Arrows indicate the multinucleated cells identified by an EYFP/CFP-Nuc double-color system. (B) Higher magnification images of A. The cell morphology was visualized by Alexa Fluor 568-conjugated phalloidin staining. Broken lines show the periphery of the EYFP/CFP-Nuc double positive syncytium. Note that CNN3-depletion induced syncytium formation without forskolin treatment and increased the number of multinucleated cells after forskolin treatment. Scale bar: 100 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

    doi: 10.1091/mbc.E10-03-0261

    Figure Lengend Snippet: CNN3 gene knockdown. (A) Control or CNN3-specific shRNA was introduced into BeWo cells expressing EYFP or CFP-Nuc. After washing, different marker expressing cells were trypsinized and mixed in the culture in the absence or presence of 50 μM forskolin for 72 h. After fixation, cells were stained with Alexa Fluor 568-conjugated phalloidin (red). Scale bar: 40 μm. Arrows indicate the multinucleated cells identified by an EYFP/CFP-Nuc double-color system. (B) Higher magnification images of A. The cell morphology was visualized by Alexa Fluor 568-conjugated phalloidin staining. Broken lines show the periphery of the EYFP/CFP-Nuc double positive syncytium. Note that CNN3-depletion induced syncytium formation without forskolin treatment and increased the number of multinucleated cells after forskolin treatment. Scale bar: 100 μm.

    Article Snippet: Differentiation was induced by treatment with 50 μM forskolin (Wako), for up to 96 h ( ; ; ) with daily exchange of the forskolin-containing medium.

    Techniques: shRNA, Expressing, Marker, Staining

    Phosphorylation-dependent association of CNN3 with actin cytoskeleton. (A) Association of ΔC CNN3 with actin. Wild-type (WT) or ΔC Flag-CNN3 expressing BeWo cells were treated with forskolin for the indicated times, and association of CNN3 and actin was analyzed by co-IP assay using anti-Flag agarose beads. Note that a significant amount of ΔC CNN3 remained in association with actin at 96h after forskolin treatment, compared with WT CNN3. (B) Subcellular localization of EYFP-CNN3. WT or ΔC EYFP-CNN3 was introduced into BeWo cells, and the localization of EYFP-CNN3 was visualized in the presence or absence of forskolin in living cells. (C) Colocalization of CNN3 and F-actin. BeWo cells expressing EYFP-CNN3 were cultivated in the presence (lower panels) or absence (upper panels) of 50 μM forskolin for 72 h. After fixation, the cells were stained by Alexa fluor 568-conjugated phalloidin (red) and DAPI (blue). Note that WT EYFP-CNN3 expressing syncytium did not colocalize with F-actin after forskolin treatment, but ΔC EYFP-CNN3 still overlapped with the F-actin even in multinucleated cells. The dotted lines show the periphery of the syncytium. Scale bar: 50 μm.

    Journal: Molecular Biology of the Cell

    Article Title: Calponin 3 Regulates Actin Cytoskeleton Rearrangement in Trophoblastic Cell Fusion

    doi: 10.1091/mbc.E10-03-0261

    Figure Lengend Snippet: Phosphorylation-dependent association of CNN3 with actin cytoskeleton. (A) Association of ΔC CNN3 with actin. Wild-type (WT) or ΔC Flag-CNN3 expressing BeWo cells were treated with forskolin for the indicated times, and association of CNN3 and actin was analyzed by co-IP assay using anti-Flag agarose beads. Note that a significant amount of ΔC CNN3 remained in association with actin at 96h after forskolin treatment, compared with WT CNN3. (B) Subcellular localization of EYFP-CNN3. WT or ΔC EYFP-CNN3 was introduced into BeWo cells, and the localization of EYFP-CNN3 was visualized in the presence or absence of forskolin in living cells. (C) Colocalization of CNN3 and F-actin. BeWo cells expressing EYFP-CNN3 were cultivated in the presence (lower panels) or absence (upper panels) of 50 μM forskolin for 72 h. After fixation, the cells were stained by Alexa fluor 568-conjugated phalloidin (red) and DAPI (blue). Note that WT EYFP-CNN3 expressing syncytium did not colocalize with F-actin after forskolin treatment, but ΔC EYFP-CNN3 still overlapped with the F-actin even in multinucleated cells. The dotted lines show the periphery of the syncytium. Scale bar: 50 μm.

    Article Snippet: Differentiation was induced by treatment with 50 μM forskolin (Wako), for up to 96 h ( ; ; ) with daily exchange of the forskolin-containing medium.

    Techniques: Expressing, Co-Immunoprecipitation Assay, Staining

    PKA C-α knockdown inhibits PGE 2 - and forskolin- induced IL-6 mRNA synthesis in human chondrocytes. T/C-28a2 chondrocytes were transfected with an small interfering RNA (siRNA) oligonucleotide sequence specific for PKA C-α before being treated with either PGE 2 (10 μM) or forskolin (20 μM) for 1 or 2 h. PKA C-α ( middle ) and IL-6 mRNA ( bottom ) expressions were determined by quantitative RT-PCR. GAPDH was served as internal control. Data are means ± SE of three independent experiments. * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Prostaglandin E2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

    doi: 10.1152/ajpcell.00508.2009

    Figure Lengend Snippet: PKA C-α knockdown inhibits PGE 2 - and forskolin- induced IL-6 mRNA synthesis in human chondrocytes. T/C-28a2 chondrocytes were transfected with an small interfering RNA (siRNA) oligonucleotide sequence specific for PKA C-α before being treated with either PGE 2 (10 μM) or forskolin (20 μM) for 1 or 2 h. PKA C-α ( middle ) and IL-6 mRNA ( bottom ) expressions were determined by quantitative RT-PCR. GAPDH was served as internal control. Data are means ± SE of three independent experiments. * P

    Article Snippet: PGE2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

    Techniques: Transfection, Small Interfering RNA, Sequencing, Quantitative RT-PCR

    Time-dependent increases of IL-6 mRNA and protein synthesis and cAMP production induced by PGE 2 or forskolin in human chondrocytes. T/C-28a2 chondrocytes or human primary articular chondrocytes ( D , H ) were treated with either PGE 2 (10 μM) ( A – D ) or forskolin (20 μM) ( E – H ) for the indicated time intervals. IL-6 mRNA expression was determined by qRT-PCR ( A , D , E , H ). GAPDH served as internal control. IL-6 protein expression levels were determined by Western blotting using an anti-IL-6 mAb ( B , F ). β-Actin was used as loading control. cAMP formation was monitored by a cAMP enzyme immunoassay kit ( C , G ). Data are means ± SE of three independent experiments, except for the experiments performed using human primary chondrocytes ( n = 1). * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Prostaglandin E2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

    doi: 10.1152/ajpcell.00508.2009

    Figure Lengend Snippet: Time-dependent increases of IL-6 mRNA and protein synthesis and cAMP production induced by PGE 2 or forskolin in human chondrocytes. T/C-28a2 chondrocytes or human primary articular chondrocytes ( D , H ) were treated with either PGE 2 (10 μM) ( A – D ) or forskolin (20 μM) ( E – H ) for the indicated time intervals. IL-6 mRNA expression was determined by qRT-PCR ( A , D , E , H ). GAPDH served as internal control. IL-6 protein expression levels were determined by Western blotting using an anti-IL-6 mAb ( B , F ). β-Actin was used as loading control. cAMP formation was monitored by a cAMP enzyme immunoassay kit ( C , G ). Data are means ± SE of three independent experiments, except for the experiments performed using human primary chondrocytes ( n = 1). * P

    Article Snippet: PGE2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay

    Dose-dependent induction of interleukin (IL)-6 mRNA synthesis by prostaglandin (PG)E 2 or forskolin in human chondrocytes. T/C-28a2 chondrocytes ( A , B ) or human primary articular chondrocytes ( C , D ) were treated with the indicated concentrations of either PGE 2 ( A , C ) or forskolin ( B , D ) for 1 h. IL-6 mRNA expression was determined by quantitative RT-PCR. GAPDH served as internal control. Data are means ± SE of three independent experiments, except for the experiments performed using human primary chondrocytes ( n = 1). * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Prostaglandin E2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

    doi: 10.1152/ajpcell.00508.2009

    Figure Lengend Snippet: Dose-dependent induction of interleukin (IL)-6 mRNA synthesis by prostaglandin (PG)E 2 or forskolin in human chondrocytes. T/C-28a2 chondrocytes ( A , B ) or human primary articular chondrocytes ( C , D ) were treated with the indicated concentrations of either PGE 2 ( A , C ) or forskolin ( B , D ) for 1 h. IL-6 mRNA expression was determined by quantitative RT-PCR. GAPDH served as internal control. Data are means ± SE of three independent experiments, except for the experiments performed using human primary chondrocytes ( n = 1). * P

    Article Snippet: PGE2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

    Techniques: Expressing, Quantitative RT-PCR

    Proposed cascade of signaling events in human T/C-28a2 chondrocytes stimulated with PGE 2 or forskolin. PGE 2 stimulates cAMP formation, which in turn upregulates PI3K/Akt and PKA activities, leading to NF-κB activation. Binding of NF-κB to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes.

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Prostaglandin E2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

    doi: 10.1152/ajpcell.00508.2009

    Figure Lengend Snippet: Proposed cascade of signaling events in human T/C-28a2 chondrocytes stimulated with PGE 2 or forskolin. PGE 2 stimulates cAMP formation, which in turn upregulates PI3K/Akt and PKA activities, leading to NF-κB activation. Binding of NF-κB to IL-6 promoter induces IL-6 synthesis in human T/C28a2 chondrocytes.

    Article Snippet: PGE2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

    Techniques: Activation Assay, Binding Assay

    PGE 2 and forskolin induce phosphorylation of Akt and cAMP responsive element binding protein (CREB) in human T/C-28a2 chondrocytes. T/C2–8a2 chondrocytes were treated with either PGE 2 (10 μM) ( A ) or forskolin (20 μM) ( B ) for the indicated periods of time. Phosphorylated Akt (Ser473), total Akt, phosphorylated CREB (Ser133) and total CREB levels are shown by immunoblotting using specific Abs. Equal loading in each lane is shown by the similar intensities of total Akt, total CREB and β-actin. Immunoblots are representative of three independent experiments, all revealing similar results. The intensity of bands was quantified relative to β-actin for each treatment using the Bio-Rad gel image system and then normalized with respect to the value obtained for the untreated control. Data are means ± SE of three independent experiments. * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Prostaglandin E2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

    doi: 10.1152/ajpcell.00508.2009

    Figure Lengend Snippet: PGE 2 and forskolin induce phosphorylation of Akt and cAMP responsive element binding protein (CREB) in human T/C-28a2 chondrocytes. T/C2–8a2 chondrocytes were treated with either PGE 2 (10 μM) ( A ) or forskolin (20 μM) ( B ) for the indicated periods of time. Phosphorylated Akt (Ser473), total Akt, phosphorylated CREB (Ser133) and total CREB levels are shown by immunoblotting using specific Abs. Equal loading in each lane is shown by the similar intensities of total Akt, total CREB and β-actin. Immunoblots are representative of three independent experiments, all revealing similar results. The intensity of bands was quantified relative to β-actin for each treatment using the Bio-Rad gel image system and then normalized with respect to the value obtained for the untreated control. Data are means ± SE of three independent experiments. * P

    Article Snippet: PGE2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

    Techniques: Binding Assay, Western Blot

    CREB1 and ATF4 are not involved in PGE 2 - and forskolin-induced IL-6 synthesis in human T/C-28a2 chondrocytes. T/C-28a2 cells were transfected with an siRNA oligonucleotide sequence specific for CREB1 or ATF4, or a control siRNA, before being treated with forskolin (20 μM) for the indicated periods of time. IL-6, CREB1, and ATF4 mRNA levels were determined by qRT-PCR. GAPDH served as internal control. Data are means ± SE of three independent experiments. * P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: Prostaglandin E2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-?B activation

    doi: 10.1152/ajpcell.00508.2009

    Figure Lengend Snippet: CREB1 and ATF4 are not involved in PGE 2 - and forskolin-induced IL-6 synthesis in human T/C-28a2 chondrocytes. T/C-28a2 cells were transfected with an siRNA oligonucleotide sequence specific for CREB1 or ATF4, or a control siRNA, before being treated with forskolin (20 μM) for the indicated periods of time. IL-6, CREB1, and ATF4 mRNA levels were determined by qRT-PCR. GAPDH served as internal control. Data are means ± SE of three independent experiments. * P

    Article Snippet: PGE2 , forskolin, the adenylate cyclase inhibitor SQ-22536, the PKA inhibitor H89, the specific activator of the exchange protein activated by cAMP (Epac) 8-(4-Chlorophenylthio)-2′- O -methyladenosine-3′,5′-cyclic monophosphate (8-pCPT-2′- O -Me-cAMP·Na or CPT), and the NF-κB inhibitor 6-amino-4-(4-phenoxyphenylethylamino)quinazoline (QNZ) were obtained from Enzo Life Sciences.

    Techniques: Transfection, Sequencing, Quantitative RT-PCR

    Pharmacologic ATR inhibition does not interfere with cAMP-induced Mitf activation SK-MEL-2 melanoma cells (n=3) (A–B) and PHMs (n=3) (C–D) were treated with 10 uM forskolin, VE821, or a combination of 10 uM forskolin and 10 uM VE-821. Whole cell lysates were collected 1 hour following treatment and immunoblotted for Mitf. B,D) Composite densitometry quantification of immunoblots (n=3 per condition) in A,C. NS denotes that no significant difference was observed between experimental groups as determined by one-way ANOVA and Tukey post hoc test. Data are expressed as mean-fold change over control ± SEM.

    Journal: Experimental dermatology

    Article Title: Divergence of cAMP signaling pathways mediating augmented nucleotide excision repair and pigment induction in melanocytes

    doi: 10.1111/exd.13291

    Figure Lengend Snippet: Pharmacologic ATR inhibition does not interfere with cAMP-induced Mitf activation SK-MEL-2 melanoma cells (n=3) (A–B) and PHMs (n=3) (C–D) were treated with 10 uM forskolin, VE821, or a combination of 10 uM forskolin and 10 uM VE-821. Whole cell lysates were collected 1 hour following treatment and immunoblotted for Mitf. B,D) Composite densitometry quantification of immunoblots (n=3 per condition) in A,C. NS denotes that no significant difference was observed between experimental groups as determined by one-way ANOVA and Tukey post hoc test. Data are expressed as mean-fold change over control ± SEM.

    Article Snippet: Forskolin (LC Laboratories) and VE-821 (Selleckchem) were utilized as indicated. siRNA targeted to ATR (Dharmacon) and MITF (Dharmacon) were performed according to the manufacturer’s instructions.

    Techniques: Inhibition, Activation Assay, Western Blot

    ATR inhibition does not affect PKA phosphorylation of CREB SK-MEL-2 melanoma cells (n=3) (A–B) and PHMs (n=3) (C–D) were treated with 10 uM forskolin, VE821, or a combination of 10 uM forskolin and 10 uM VE-821. Whole cell lysates were collected 1 hour following treatment and immunoblotted for pSer133-CREB. B,D) Composite densitometry quantification of immunoblots (n=3 per condition) in A,C. Values not sharing a common letter are significantly different as determined by one-way ANOVA and Tukey post hoc test (p

    Journal: Experimental dermatology

    Article Title: Divergence of cAMP signaling pathways mediating augmented nucleotide excision repair and pigment induction in melanocytes

    doi: 10.1111/exd.13291

    Figure Lengend Snippet: ATR inhibition does not affect PKA phosphorylation of CREB SK-MEL-2 melanoma cells (n=3) (A–B) and PHMs (n=3) (C–D) were treated with 10 uM forskolin, VE821, or a combination of 10 uM forskolin and 10 uM VE-821. Whole cell lysates were collected 1 hour following treatment and immunoblotted for pSer133-CREB. B,D) Composite densitometry quantification of immunoblots (n=3 per condition) in A,C. Values not sharing a common letter are significantly different as determined by one-way ANOVA and Tukey post hoc test (p

    Article Snippet: Forskolin (LC Laboratories) and VE-821 (Selleckchem) were utilized as indicated. siRNA targeted to ATR (Dharmacon) and MITF (Dharmacon) were performed according to the manufacturer’s instructions.

    Techniques: Inhibition, Western Blot

    MITF inhibition does not affect PKA-mediated generation of pS435-ATR and has no functional impact on melanocyte nucleotide excision repair (NER) (A,B) SK-MEL-2 melanoma cells (n=3) (A,C) and primary human melanocytes (PHMs; n=4) (B,D) were treated with scrambled siRNA or siRNA directed to MITF prior to treatment with 10 uM forskolin. Whole cell lysates were collected at 1 hour and pSer435-ATR levels were determined by kinase assay (A,B). Values not sharing a common letter are significantly different as determined by one-way ANOVA and Tukey post-hoc test (p

    Journal: Experimental dermatology

    Article Title: Divergence of cAMP signaling pathways mediating augmented nucleotide excision repair and pigment induction in melanocytes

    doi: 10.1111/exd.13291

    Figure Lengend Snippet: MITF inhibition does not affect PKA-mediated generation of pS435-ATR and has no functional impact on melanocyte nucleotide excision repair (NER) (A,B) SK-MEL-2 melanoma cells (n=3) (A,C) and primary human melanocytes (PHMs; n=4) (B,D) were treated with scrambled siRNA or siRNA directed to MITF prior to treatment with 10 uM forskolin. Whole cell lysates were collected at 1 hour and pSer435-ATR levels were determined by kinase assay (A,B). Values not sharing a common letter are significantly different as determined by one-way ANOVA and Tukey post-hoc test (p

    Article Snippet: Forskolin (LC Laboratories) and VE-821 (Selleckchem) were utilized as indicated. siRNA targeted to ATR (Dharmacon) and MITF (Dharmacon) were performed according to the manufacturer’s instructions.

    Techniques: Inhibition, Functional Assay, Kinase Assay

    ATR inhibition does not interfere with cAMP-induced pigment induction SK-MEL-2 melanoma cells (n=3) (A,C) and primary human melanocytes (PHMs; n=4) (B,D) were treated with vehicle controls 10 uM VE-821 or 10 uM forskolin. Cells were collected, incubated with L-DOPA (see methods) and pelleted after 2h. A,B) photographs of microcentrifuge tubes showing cell pellets of indicated conditions. C,D) Cells were lysed in soluene-350 solution and spectrophotometry of supernatants was quantified at 492 nm. Values not sharing a common letter are significantly different as determined by one-way ANOVA and Tukey post hoc test (p

    Journal: Experimental dermatology

    Article Title: Divergence of cAMP signaling pathways mediating augmented nucleotide excision repair and pigment induction in melanocytes

    doi: 10.1111/exd.13291

    Figure Lengend Snippet: ATR inhibition does not interfere with cAMP-induced pigment induction SK-MEL-2 melanoma cells (n=3) (A,C) and primary human melanocytes (PHMs; n=4) (B,D) were treated with vehicle controls 10 uM VE-821 or 10 uM forskolin. Cells were collected, incubated with L-DOPA (see methods) and pelleted after 2h. A,B) photographs of microcentrifuge tubes showing cell pellets of indicated conditions. C,D) Cells were lysed in soluene-350 solution and spectrophotometry of supernatants was quantified at 492 nm. Values not sharing a common letter are significantly different as determined by one-way ANOVA and Tukey post hoc test (p

    Article Snippet: Forskolin (LC Laboratories) and VE-821 (Selleckchem) were utilized as indicated. siRNA targeted to ATR (Dharmacon) and MITF (Dharmacon) were performed according to the manufacturer’s instructions.

    Techniques: Inhibition, Incubation, Spectrophotometry

    Target dependent effects of miR-449a on proliferation, differentiation and glioma biology ( a ) in stemness—maintaining growth conditions with EGF, FGF enriched serum-free medium, miR-449a—mediated effects are predominantly exerted through CCND1 inhibition, thus reducing invasion and proliferation. For example, miR-449a high Rb/p53 cells have low expression levels of Ccdn1, proliferate and migrate slower, and have less self-renewal capacity. b in growth conditions promoting neural phenotypes in vitro, such as FBS, Forskolin, retinoic acid and in vivo experimental settings and humans gliomas, miR-449a suppresses GPR158, reducing neural marker expression, and is associated with higher glioma grade and shorter survival. Numbers and letters provide a reference to figures in the text

    Journal: Oncogene

    Article Title: Inhibition of GPR158 by microRNA-449a suppresses neural lineage of glioma stem/progenitor cells and correlates with higher glioma grades

    doi: 10.1038/s41388-018-0277-1

    Figure Lengend Snippet: Target dependent effects of miR-449a on proliferation, differentiation and glioma biology ( a ) in stemness—maintaining growth conditions with EGF, FGF enriched serum-free medium, miR-449a—mediated effects are predominantly exerted through CCND1 inhibition, thus reducing invasion and proliferation. For example, miR-449a high Rb/p53 cells have low expression levels of Ccdn1, proliferate and migrate slower, and have less self-renewal capacity. b in growth conditions promoting neural phenotypes in vitro, such as FBS, Forskolin, retinoic acid and in vivo experimental settings and humans gliomas, miR-449a suppresses GPR158, reducing neural marker expression, and is associated with higher glioma grade and shorter survival. Numbers and letters provide a reference to figures in the text

    Article Snippet: 24 h after transfection/transduction growth medium was exchanged with differentiation medium containing DMEM/F12, 2% B27, 1% penicillin-streptomycin, and supplemented with 3% FBS, or 10 uM RA (Sigma, R2625) and 20 uM Forskolin (Abcam, ab120058) for neuronal differentiation, or with 50 ng/ml LIF (Santa Cruz, sc-4989) and 50 ng/ml BMP2 (Thermo Fisher, PHC7145) for astrocytic differentiation [ ].

    Techniques: Inhibition, Expressing, In Vitro, In Vivo, Marker

    Immunoblot analysis of protein kinase A (PKA) substrates in T cells. (A) HuT78 cells were serum starved for 4 h and then treated with DMSO ( control ) or 30 µM forskolin for 30 min and lysed. Protein lysates were immunoprecipitated (IP) using either phospho-PKA substrate antibody ( p-PKA substrate ). (B) Control and CG-NAP knockdown ( KD ) HuT78 T-cells were either unstimulated or LFA-1-stimulated (+rICAM-1) for 1 h, lysed and IP using anti-PKARIIα antibody. Immunoprecipitates were resolved on SDS-PAGE and subjected to Western blotting with anti-CG-NAP, pericentrin, dynein, or PKARIIα antibodies. (C) Control or CG-NAP KD HuT78 cells were treated with 30 µM forskolin for 30 min and protein lysates were IP using either phospho-PKA substrate antibody or control IgG. Immunoprecipitates were resolved on SDS-PAGE and subjected to Western blotting with anti-pericentrin and anti-dynein antibodies. Whole cell lysates ( WCL , 10 µg each) were used as input controls for Western immunoblots; gel lanes indicated by “X” are empty lanes, i.e., no protein loaded. Relative densitometry graph of Western immunoblots obtained from at least three independent experiments are presented (mean ± SEM, * p

    Journal: Frontiers in Immunology

    Article Title: Centrosome- and Golgi-Localized Protein Kinase N-Associated Protein Serves As a Docking Platform for Protein Kinase A Signaling and Microtubule Nucleation in Migrating T-Cells

    doi: 10.3389/fimmu.2018.00397

    Figure Lengend Snippet: Immunoblot analysis of protein kinase A (PKA) substrates in T cells. (A) HuT78 cells were serum starved for 4 h and then treated with DMSO ( control ) or 30 µM forskolin for 30 min and lysed. Protein lysates were immunoprecipitated (IP) using either phospho-PKA substrate antibody ( p-PKA substrate ). (B) Control and CG-NAP knockdown ( KD ) HuT78 T-cells were either unstimulated or LFA-1-stimulated (+rICAM-1) for 1 h, lysed and IP using anti-PKARIIα antibody. Immunoprecipitates were resolved on SDS-PAGE and subjected to Western blotting with anti-CG-NAP, pericentrin, dynein, or PKARIIα antibodies. (C) Control or CG-NAP KD HuT78 cells were treated with 30 µM forskolin for 30 min and protein lysates were IP using either phospho-PKA substrate antibody or control IgG. Immunoprecipitates were resolved on SDS-PAGE and subjected to Western blotting with anti-pericentrin and anti-dynein antibodies. Whole cell lysates ( WCL , 10 µg each) were used as input controls for Western immunoblots; gel lanes indicated by “X” are empty lanes, i.e., no protein loaded. Relative densitometry graph of Western immunoblots obtained from at least three independent experiments are presented (mean ± SEM, * p

    Article Snippet: Phospho-PKA substrate (RRXS*/T*) rabbit monoclonal antibody, rabbit polyclonal anti-acetylated α-tubulin antibody, and forskolin were from Cell Signaling Technology.

    Techniques: Immunoprecipitation, SDS Page, Western Blot

    Glucocorticoid receptor (GR) expression in CEM and MM.1 cells. (A,B,E) Quantitative real-time PCR analysis of GR mRNA expression in CEM-S2 (A) , CEM-R8 (B) , and MM.1S (E) cells, following treatment with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM forskolin (R+F), or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+D) at different times as indicated. Data represent the mean ± SD of at least two independent experiments assayed in triplicate, shown as fold change relative to control. (C,D,F) Western blot analysis of GR protein expression in CEM-S2 (C) , CEM-R8 (D) , and MM.1S (F) cells following treatment at different times with dexamethasone and/or rolipram and forskolin as indicated. Equal amounts of protein (20 μg) from whole cell lysates were added per lane. Data shown represents one of at least two independent experiments with similar results. The numbers at the top of the Western blot images represent the GR band densities following different treatments relative to control, normalized based on the density of the GAPDH housekeeping protein, calculated as described in Section “Materials and Methods.”

    Journal: Frontiers in Pharmacology

    Article Title: Effect of cAMP signaling on expression of glucocorticoid receptor, Bim and Bad in glucocorticoid-sensitive and resistant leukemic and multiple myeloma cells

    doi: 10.3389/fphar.2015.00230

    Figure Lengend Snippet: Glucocorticoid receptor (GR) expression in CEM and MM.1 cells. (A,B,E) Quantitative real-time PCR analysis of GR mRNA expression in CEM-S2 (A) , CEM-R8 (B) , and MM.1S (E) cells, following treatment with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM forskolin (R+F), or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+D) at different times as indicated. Data represent the mean ± SD of at least two independent experiments assayed in triplicate, shown as fold change relative to control. (C,D,F) Western blot analysis of GR protein expression in CEM-S2 (C) , CEM-R8 (D) , and MM.1S (F) cells following treatment at different times with dexamethasone and/or rolipram and forskolin as indicated. Equal amounts of protein (20 μg) from whole cell lysates were added per lane. Data shown represents one of at least two independent experiments with similar results. The numbers at the top of the Western blot images represent the GR band densities following different treatments relative to control, normalized based on the density of the GAPDH housekeeping protein, calculated as described in Section “Materials and Methods.”

    Article Snippet: Materials Dexamethasone, hydrocortisone, rolipram, forskolin, and 1,9-dideoxyforskolin were obtained from Biomol (Plymouth Meeting, PA, USA).

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

    Effect of 1,9-dideoxyforskolin on CEM cells. Cell viability of CEM-S2 cells (A) and CEM-R8 cells (B) . Cells were treated with 10 μM forskolin (Fsk) or 10 μM 1,9-dideoxyforskolin (dFsk) in the presence or absence of 10 μM hydrocortisone (HC) for 72 h. After treatment, cell viability was determined by MTS assay. Data represent the mean ± SD of at least two independent experiments assayed in triplicate.

    Journal: Frontiers in Pharmacology

    Article Title: Effect of cAMP signaling on expression of glucocorticoid receptor, Bim and Bad in glucocorticoid-sensitive and resistant leukemic and multiple myeloma cells

    doi: 10.3389/fphar.2015.00230

    Figure Lengend Snippet: Effect of 1,9-dideoxyforskolin on CEM cells. Cell viability of CEM-S2 cells (A) and CEM-R8 cells (B) . Cells were treated with 10 μM forskolin (Fsk) or 10 μM 1,9-dideoxyforskolin (dFsk) in the presence or absence of 10 μM hydrocortisone (HC) for 72 h. After treatment, cell viability was determined by MTS assay. Data represent the mean ± SD of at least two independent experiments assayed in triplicate.

    Article Snippet: Materials Dexamethasone, hydrocortisone, rolipram, forskolin, and 1,9-dideoxyforskolin were obtained from Biomol (Plymouth Meeting, PA, USA).

    Techniques: MTS Assay

    Bim expression in CEM and MM.1 cells. (A) Quantitative real-time PCR analysis of Bim mRNA expression in CEM-S2 cells (top), at different times following treatment with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM forskolin (R+F) or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+D) as indicated. Data represent the mean ± SD of at least two independent experiments assayed in triplicate, shown as fold change relative to control. (bottom), Western blot analysis of expression of Bim protein. Cells were treated at different times with dexamethasone and/or rolipram and forskolin as indicated, and Bim protein expression was determined by immunoblots of whole cell lysates. Equal amounts of protein (20 μg) were loaded per lane. Data shown represents one of at least two independent experiments with similar results. The numbers at the top of the Western blot images represent the BimEL band densities following different treatments relative to control, normalized based on the density of the GAPDH housekeeping protein, calculated as described in Section “Materials and Methods.” (B) CEM-R8 cell Bim mRNA (top) and protein (bottom) expression. (C) Bim mRNA (top) and protein (bottom) expression in MM.1S cells. (D) Bim mRNA (top) and protein (bottom) expression in MM.1R cells. Conditions were as in (A) .

    Journal: Frontiers in Pharmacology

    Article Title: Effect of cAMP signaling on expression of glucocorticoid receptor, Bim and Bad in glucocorticoid-sensitive and resistant leukemic and multiple myeloma cells

    doi: 10.3389/fphar.2015.00230

    Figure Lengend Snippet: Bim expression in CEM and MM.1 cells. (A) Quantitative real-time PCR analysis of Bim mRNA expression in CEM-S2 cells (top), at different times following treatment with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM forskolin (R+F) or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+D) as indicated. Data represent the mean ± SD of at least two independent experiments assayed in triplicate, shown as fold change relative to control. (bottom), Western blot analysis of expression of Bim protein. Cells were treated at different times with dexamethasone and/or rolipram and forskolin as indicated, and Bim protein expression was determined by immunoblots of whole cell lysates. Equal amounts of protein (20 μg) were loaded per lane. Data shown represents one of at least two independent experiments with similar results. The numbers at the top of the Western blot images represent the BimEL band densities following different treatments relative to control, normalized based on the density of the GAPDH housekeeping protein, calculated as described in Section “Materials and Methods.” (B) CEM-R8 cell Bim mRNA (top) and protein (bottom) expression. (C) Bim mRNA (top) and protein (bottom) expression in MM.1S cells. (D) Bim mRNA (top) and protein (bottom) expression in MM.1R cells. Conditions were as in (A) .

    Article Snippet: Materials Dexamethasone, hydrocortisone, rolipram, forskolin, and 1,9-dideoxyforskolin were obtained from Biomol (Plymouth Meeting, PA, USA).

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

    Glucocorticoid receptor alpha (GR) promoter 1A, 1B, and 1C expression in CEM and MM.1 cells. Quantitative real-time PCR analysis of GR1A3, 1B and 1C mRNA expression in both CEM and MM.1 cells. (A) , GR1A3(i), 1B(ii), and 1C(iii) expression in CEM-S2 cells. (B) , GR1A3(i), 1B(ii), and 1C(iii) expression in CEM-R8 cells. (C) , GR1A3(i), 1B(ii), and 1C(iii) expression in MM.1S cells. Points were taken following treatment with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM forskolin (R+F), or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+D) for different times as indicated. Data represent the mean ± SD of at least two independent experiments assayed in triplicate.

    Journal: Frontiers in Pharmacology

    Article Title: Effect of cAMP signaling on expression of glucocorticoid receptor, Bim and Bad in glucocorticoid-sensitive and resistant leukemic and multiple myeloma cells

    doi: 10.3389/fphar.2015.00230

    Figure Lengend Snippet: Glucocorticoid receptor alpha (GR) promoter 1A, 1B, and 1C expression in CEM and MM.1 cells. Quantitative real-time PCR analysis of GR1A3, 1B and 1C mRNA expression in both CEM and MM.1 cells. (A) , GR1A3(i), 1B(ii), and 1C(iii) expression in CEM-S2 cells. (B) , GR1A3(i), 1B(ii), and 1C(iii) expression in CEM-R8 cells. (C) , GR1A3(i), 1B(ii), and 1C(iii) expression in MM.1S cells. Points were taken following treatment with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM forskolin (R+F), or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+D) for different times as indicated. Data represent the mean ± SD of at least two independent experiments assayed in triplicate.

    Article Snippet: Materials Dexamethasone, hydrocortisone, rolipram, forskolin, and 1,9-dideoxyforskolin were obtained from Biomol (Plymouth Meeting, PA, USA).

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Bad expression in CEM and MM.1 cells. Top panels: quantitative real-time PCR analysis of Bad mRNA at different times after treatment with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM forskolin (R+F), or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+D) as indicated. Data represent the mean ± SD of at least two independent experiments assayed in triplicate, shown as fold change relative to control. Bottom panels: Western blot analysis of expression of total Bad and p-Bad (ser112) protein. Cells were treated at different times with dexamethasone and/or rolipram and forskolin as indicated, and Bad protein expression was determined by immunoblots of whole cell lysates. An equal amount of protein (20 μg) was loaded per lane. Densities of phosphorylated Bad (P) and total Bad (T) are given at the top of the Western blot panels, calculated as described in Section “Materials and Methods.” Data shown represents one of at least two independent experiments with similar results. (A) CEM-S2 cells; (B) CEM-R8 cells; (C) MM.1S cells, and (D) MM.1R cells.

    Journal: Frontiers in Pharmacology

    Article Title: Effect of cAMP signaling on expression of glucocorticoid receptor, Bim and Bad in glucocorticoid-sensitive and resistant leukemic and multiple myeloma cells

    doi: 10.3389/fphar.2015.00230

    Figure Lengend Snippet: Bad expression in CEM and MM.1 cells. Top panels: quantitative real-time PCR analysis of Bad mRNA at different times after treatment with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM forskolin (R+F), or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+D) as indicated. Data represent the mean ± SD of at least two independent experiments assayed in triplicate, shown as fold change relative to control. Bottom panels: Western blot analysis of expression of total Bad and p-Bad (ser112) protein. Cells were treated at different times with dexamethasone and/or rolipram and forskolin as indicated, and Bad protein expression was determined by immunoblots of whole cell lysates. An equal amount of protein (20 μg) was loaded per lane. Densities of phosphorylated Bad (P) and total Bad (T) are given at the top of the Western blot panels, calculated as described in Section “Materials and Methods.” Data shown represents one of at least two independent experiments with similar results. (A) CEM-S2 cells; (B) CEM-R8 cells; (C) MM.1S cells, and (D) MM.1R cells.

    Article Snippet: Materials Dexamethasone, hydrocortisone, rolipram, forskolin, and 1,9-dideoxyforskolin were obtained from Biomol (Plymouth Meeting, PA, USA).

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

    Effect of dexamethasone, rolipram, and forskolin on viability of glucocorticoid-sensitive and glucocorticoid-resistant CEM and MM.1 cells. (A) CEM-S2 cells, (B) CEM-R8 cells, (C) MM.1S cells, and (D) MM.1R cells were treated with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM forskolin (R+F), or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+Dex) for 72 h. Cell viability was then determined by the MTS method. Data represent the mean ± SD of at least two independent experiments assayed in triplicate.

    Journal: Frontiers in Pharmacology

    Article Title: Effect of cAMP signaling on expression of glucocorticoid receptor, Bim and Bad in glucocorticoid-sensitive and resistant leukemic and multiple myeloma cells

    doi: 10.3389/fphar.2015.00230

    Figure Lengend Snippet: Effect of dexamethasone, rolipram, and forskolin on viability of glucocorticoid-sensitive and glucocorticoid-resistant CEM and MM.1 cells. (A) CEM-S2 cells, (B) CEM-R8 cells, (C) MM.1S cells, and (D) MM.1R cells were treated with 1 μM dexamethasone (Dex), 10 μM rolipram plus 10 μM forskolin (R+F), or 1 μM dexamethasone plus 10 μM rolipram plus 10 μM forskolin (R+F+Dex) for 72 h. Cell viability was then determined by the MTS method. Data represent the mean ± SD of at least two independent experiments assayed in triplicate.

    Article Snippet: Materials Dexamethasone, hydrocortisone, rolipram, forskolin, and 1,9-dideoxyforskolin were obtained from Biomol (Plymouth Meeting, PA, USA).

    Techniques:

    Corrector treatment increases CFTR activation response of nonsense variants in exon 22 that result in mature truncated CFTR. (A) Schematic of CFTR-Expression Minigene with abridged introns 21 and abridged intron 22 (EMG-i21-i22) constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and two abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (B) RT-qPCR showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Steady state levels of CFTR protein from HEK293 cells transiently transfected with wild-type EMG or EMGs with different nonsense variants. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody-MM13-4 (EMD Millipore). (D) Representative IB showing sensitivity of CFTR to PNGaseF and Endo H. Mature complex glycosylated band is sensitive to PNGase only, whereas immature core glycosylated band is sensitive to both PNGase and EndoH. (E). Schematic illustration of the nonsense variants in the protein context showing their classification into two groups based on mRNA stability and protein maturity. Each nonsense variant truncates CFTR at intracellular loop 6 (ICL6) just before NBD2. (F) A representative Ussing chamber tracing of CFBE cells stably expressing S1196X-EMG. Short-circuit (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. ( G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated I sc without modulator treatment in cells expressing EMG i21-i22. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001), n.s. (not significant, P > 0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.

    Journal: PLoS Genetics

    Article Title: Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

    doi: 10.1371/journal.pgen.1007723

    Figure Lengend Snippet: Corrector treatment increases CFTR activation response of nonsense variants in exon 22 that result in mature truncated CFTR. (A) Schematic of CFTR-Expression Minigene with abridged introns 21 and abridged intron 22 (EMG-i21-i22) constructed in pcDNA5FRT plasmid. CFTR exons are shown in boxes and two abridged introns in dashed lines. The location of each studied variant is shown relative to the CFTR exons and regions predicted to elicit NMD. (B) RT-qPCR showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with truncations at residue position, as indicated on the labels. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Steady state levels of CFTR protein from HEK293 cells transiently transfected with wild-type EMG or EMGs with different nonsense variants. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody-MM13-4 (EMD Millipore). (D) Representative IB showing sensitivity of CFTR to PNGaseF and Endo H. Mature complex glycosylated band is sensitive to PNGase only, whereas immature core glycosylated band is sensitive to both PNGase and EndoH. (E). Schematic illustration of the nonsense variants in the protein context showing their classification into two groups based on mRNA stability and protein maturity. Each nonsense variant truncates CFTR at intracellular loop 6 (ICL6) just before NBD2. (F) A representative Ussing chamber tracing of CFBE cells stably expressing S1196X-EMG. Short-circuit (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. ( G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated I sc without modulator treatment in cells expressing EMG i21-i22. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001), n.s. (not significant, P > 0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.

    Article Snippet: After stabilization of transepithelial current, 10 μM forskolin (Selleckchem) was added to the basolateral chamber to stimulate generation of cAMP and activation of CFTR, followed by administration of 10 μM CFTR inhibitor-172 (Selleckchem) in the apical chamber to block CFTR-mediated currents.

    Techniques: Activation Assay, Expressing, Construct, Plasmid Preparation, Variant Assay, Quantitative RT-PCR, Transfection, Stable Transfection

    NMD inhibition has a synergistic effect on corrector-potentiator combination response in stable cells expressing nonsense variants in exon 22 that produce mature truncated CFTR. (A) Relative expression of the alternate CFTR allele in the primary nasal cells of CF individuals carrying exon 22 nonsense variant. Pyrosequencing assay was designed such that exon 22 with upstream and downstream flanking exons was amplified from the corresponding cDNA preparations. Sequencing primer yielded relative abundances of alternate alleles at the respective loci where nucleotide change occurred. (B ) CFTR mRNA decay in HEK293 cells stably expressing wild type EMG or EMG harboring nonsense variants R1158X or S1196X. Actinomycin D (3 μg/ml) was added at time 0 to induce transcriptional shut-down. Cells were collected at the indicated time points. Levels of the CFTR mRNAs were assessed by RT-qPCR, normalized to B2M mRNA and displayed as a percentage of the levels at t = 0. Mean ± SEM ( n = 3) (C) Efficiency of siRNA mediated knock down of UPF1 detected on IB of whole cell lysates collected from HEK293 cells stably expressing either R1158X or S1196X. GAPDH siRNA and non-target (NT) siRNA were used as positive and negative controls respectively. Beta-Actin was used as loading control. (D) Effect of direct NMD inhibition on the level of CFTR transcript by siRNA mediated knock down of UPF1 in HEK293 cells stably expressing either R1158X or S1196X. Levels of the CFTR mRNAs were assessed by RT-qPCR and normalized to B2M mRNA. Mean ± SEM, n = 3 independent biological triplicates, P value was determined by two way ANOVA. ** ( P ≤0.01) and *** ( P ≤0.001) indicate significant difference when compared with CFTR mRNA abundance in untreated cells (E) Short-circuit (I sc ) tracings of CFBE-S1196X stable cells recorded in Ussing chambers after direct inhibition of NMD by UPF1 . Cells were transfected with Upf1 siRNA at 50% confluency for 4 days before being mounted on Ussing chambers. GAPDH and non-targeted (NT) siRNA transfections were used as controls. Cells were incubated with lumacaftor (3 μM) or DMSO (0.03%) during last 48h of siRNA transfections. (F) Stacked bar graphs indicate effect of UPF1 siRNA in combination with CFTR modulators. Change in I sc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3). P value was determined by one way ANOVA. ** ( P ≤0.01) indicate significant difference when compared with forskolin stimulated CFTR function in NT siRNA transfected cells and **** ( P ≤0.0001) indicate significant difference when compared with ivacaftor activated CFTR function in NT siRNA transfected cells incubated with or without lumacaftor. WT-CFTR function represents forskolin stimulated ΔI sc without modulator treatment in cells expressing EMG i21-i22.

    Journal: PLoS Genetics

    Article Title: Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

    doi: 10.1371/journal.pgen.1007723

    Figure Lengend Snippet: NMD inhibition has a synergistic effect on corrector-potentiator combination response in stable cells expressing nonsense variants in exon 22 that produce mature truncated CFTR. (A) Relative expression of the alternate CFTR allele in the primary nasal cells of CF individuals carrying exon 22 nonsense variant. Pyrosequencing assay was designed such that exon 22 with upstream and downstream flanking exons was amplified from the corresponding cDNA preparations. Sequencing primer yielded relative abundances of alternate alleles at the respective loci where nucleotide change occurred. (B ) CFTR mRNA decay in HEK293 cells stably expressing wild type EMG or EMG harboring nonsense variants R1158X or S1196X. Actinomycin D (3 μg/ml) was added at time 0 to induce transcriptional shut-down. Cells were collected at the indicated time points. Levels of the CFTR mRNAs were assessed by RT-qPCR, normalized to B2M mRNA and displayed as a percentage of the levels at t = 0. Mean ± SEM ( n = 3) (C) Efficiency of siRNA mediated knock down of UPF1 detected on IB of whole cell lysates collected from HEK293 cells stably expressing either R1158X or S1196X. GAPDH siRNA and non-target (NT) siRNA were used as positive and negative controls respectively. Beta-Actin was used as loading control. (D) Effect of direct NMD inhibition on the level of CFTR transcript by siRNA mediated knock down of UPF1 in HEK293 cells stably expressing either R1158X or S1196X. Levels of the CFTR mRNAs were assessed by RT-qPCR and normalized to B2M mRNA. Mean ± SEM, n = 3 independent biological triplicates, P value was determined by two way ANOVA. ** ( P ≤0.01) and *** ( P ≤0.001) indicate significant difference when compared with CFTR mRNA abundance in untreated cells (E) Short-circuit (I sc ) tracings of CFBE-S1196X stable cells recorded in Ussing chambers after direct inhibition of NMD by UPF1 . Cells were transfected with Upf1 siRNA at 50% confluency for 4 days before being mounted on Ussing chambers. GAPDH and non-targeted (NT) siRNA transfections were used as controls. Cells were incubated with lumacaftor (3 μM) or DMSO (0.03%) during last 48h of siRNA transfections. (F) Stacked bar graphs indicate effect of UPF1 siRNA in combination with CFTR modulators. Change in I sc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3). P value was determined by one way ANOVA. ** ( P ≤0.01) indicate significant difference when compared with forskolin stimulated CFTR function in NT siRNA transfected cells and **** ( P ≤0.0001) indicate significant difference when compared with ivacaftor activated CFTR function in NT siRNA transfected cells incubated with or without lumacaftor. WT-CFTR function represents forskolin stimulated ΔI sc without modulator treatment in cells expressing EMG i21-i22.

    Article Snippet: After stabilization of transepithelial current, 10 μM forskolin (Selleckchem) was added to the basolateral chamber to stimulate generation of cAMP and activation of CFTR, followed by administration of 10 μM CFTR inhibitor-172 (Selleckchem) in the apical chamber to block CFTR-mediated currents.

    Techniques: Inhibition, Expressing, Variant Assay, Pyrosequencing Assay, Amplification, Sequencing, Stable Transfection, Quantitative RT-PCR, Transfection, Incubation

    Nonsense and frameshift mutations at 3’ region that synthesize complex glycosylated truncated protein can respond to CFTR modulators. (A) Schematic of CFTR-Expression Minigene with full-length introns 25 and 26 (EMG-i25-i26) constructed in pcDNA5FRT plasmid. CFTR expression is driven by a CMV promoter. The location of each studied variant is shown relative to CFTR exons and regions predicted to elicit NMD. (B) Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with nonsense or frameshift variants, as indicated. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Immunoblot (IB) of the steady state amounts of immature core-glycosylated ( band B ) and the mature complex-glycosylated mature CFTR protein ( band C ). Lysates were collected from HEK293 cells expressing WT-EMG or EMGs with different PTC-generating variants. Lysates from cells expressing either intronless WT CFTR or F508del served as controls, or empty vector as negative control. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody (596 # Cystic Fibrosis Foundation Therapeutics). (D) Schematic illustration showing three groups of 3’- nonsense variants based on mRNA stability and protein maturity. (E) Immunoblot of HEK293 stable cells expressing Q1390X or E1418X. The cells were incubated for 48 h with DMSO (.03%) or corrector compounds (lumacaftor and tezacaftor either alone or in combination—3 μM each). CFTR was visualized with anti-CFTR antibody, 596 (CFFT). (F) A representative Ussing chamber tracing of EMG E1418X-expressing CFBE stable cells grown on snap-wells. Short-circuit current (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. Cells were mounted on Ussing chambers to measure CFTR mediated chloride channel activity as a proxy of CFTR function. After stabilization of the basal current, forskolin (10 μM) was added to the basolateral chamber followed by potentiator, ivacaftor (10 μM), and CFTR Inhibitor 172 (10 μM) added to the apical chambers. Inh-172 was added earlier in DMSO no ivacaftor (dashed blue line) treated cells. (G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i25-i26. P value was determined by one way ANOVA. **** P ≤0.0001, ** P ≤0.01, * P ≤0.05, and n.s. (not significant, P > 0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.

    Journal: PLoS Genetics

    Article Title: Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

    doi: 10.1371/journal.pgen.1007723

    Figure Lengend Snippet: Nonsense and frameshift mutations at 3’ region that synthesize complex glycosylated truncated protein can respond to CFTR modulators. (A) Schematic of CFTR-Expression Minigene with full-length introns 25 and 26 (EMG-i25-i26) constructed in pcDNA5FRT plasmid. CFTR expression is driven by a CMV promoter. The location of each studied variant is shown relative to CFTR exons and regions predicted to elicit NMD. (B) Real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) showing relative steady state levels of CFTR transcript in HEK293 stable cells expressing wild-type EMG or EMGs with nonsense or frameshift variants, as indicated. Values were normalized to B2M . Mean ± SEM ( n = 3) measured in triplicates. P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with CFTR mRNA abundance in cells expressing WT-EMG. (C) Immunoblot (IB) of the steady state amounts of immature core-glycosylated ( band B ) and the mature complex-glycosylated mature CFTR protein ( band C ). Lysates were collected from HEK293 cells expressing WT-EMG or EMGs with different PTC-generating variants. Lysates from cells expressing either intronless WT CFTR or F508del served as controls, or empty vector as negative control. 40 μg of total cell lysates were electrophoresed and IB was probed with anti-CFTR antibody (596 # Cystic Fibrosis Foundation Therapeutics). (D) Schematic illustration showing three groups of 3’- nonsense variants based on mRNA stability and protein maturity. (E) Immunoblot of HEK293 stable cells expressing Q1390X or E1418X. The cells were incubated for 48 h with DMSO (.03%) or corrector compounds (lumacaftor and tezacaftor either alone or in combination—3 μM each). CFTR was visualized with anti-CFTR antibody, 596 (CFFT). (F) A representative Ussing chamber tracing of EMG E1418X-expressing CFBE stable cells grown on snap-wells. Short-circuit current (I sc ) measurements were recorded in Ussing chambers after treatment of cells with 0.03% DMSO (vehicle) or 3 μM corrector compounds (lumacaftor/tezacaftor or both) for 48 h. Cells were mounted on Ussing chambers to measure CFTR mediated chloride channel activity as a proxy of CFTR function. After stabilization of the basal current, forskolin (10 μM) was added to the basolateral chamber followed by potentiator, ivacaftor (10 μM), and CFTR Inhibitor 172 (10 μM) added to the apical chambers. Inh-172 was added earlier in DMSO no ivacaftor (dashed blue line) treated cells. (G and H) Stacked bar graphs indicate effect of modulator treatment on CFBE (G) and MDCK (H) stable cells expressing different CFTR 3’ nonsense variants. Change in I sc (ΔIsc) was defined as the current inhibited by Inh-172 after sustained Isc responses were achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Mean ± SEM ( n = 3–8). WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i25-i26. P value was determined by one way ANOVA. **** P ≤0.0001, ** P ≤0.01, * P ≤0.05, and n.s. (not significant, P > 0.05); when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells expressing respective variant.

    Article Snippet: After stabilization of transepithelial current, 10 μM forskolin (Selleckchem) was added to the basolateral chamber to stimulate generation of cAMP and activation of CFTR, followed by administration of 10 μM CFTR inhibitor-172 (Selleckchem) in the apical chamber to block CFTR-mediated currents.

    Techniques: Expressing, Construct, Plasmid Preparation, Variant Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Negative Control, Incubation, Activity Assay

    CFTR nonsense variant with splicing defect has residual function that benefits from modulator treatments. (A) A schematic illustration of CFTR-Expression Minigene with introns 14–18 (introns 14 and 16 are full-length, and 15, 17, 18 are abridged). Arrow indicates location of E831X (top). CFTR mRNA splicing patterns of the total RNA extracted from HEK293 cells transiently transfected with E831X-EMG. (B) Steady state amounts of different isoforms of CFTR produced from E831X-EMG-i14-i18 expressed transiently in HEK293 cells. Lysates from cells expressing WT-EMG i14-i18, intronless WT CFTR or F508del served as positive controls, and empty vector as negative control. Immunoblot (IB) was probed with anti-CFTR antibody, 596 (CFFT). Horizontal arrows indicate to isoforms corresponding to (i) a normal codon (E831) substituted with a stop codon, (ii) deletion of complete exon 16, and (iii) deletion of a single amino acid E831. Beta-Actin was used as loading control. (C) Short-circuit (I sc ) tracing of CFTR function observed in CFBE-stable cells expressing E831X mounted on Ussing chamber. Cells were treated for 48 h with correctors (lumacaftor/tezacaftor or both, 3 μM each) and acutely with potentiator (ivacaftor, 10 μM). Change in I sc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data are presented as mean±SEM (n = 3). P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells. WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i14-i18. (D ) A tracing of CFTR function observed in primary nasal epithelial cells of an individual harboring E831X/F508del. CF-Human nasal epithelial (HNE) cells were treated for 24 h with lumacaftor and tezacaftor, 3 μM each, and acutely with Ivacaftor (10 μM). Stacked bar graph is a comparison of improvement in CFTR function of E831X/F508del vs F508del/Indel. Alternate Indel alleles were either 2184insA, 2183delAA > G, or 3659del C. P value was determined by one way ANOVA. ** indicates significant difference ( P ≤0.01) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated E831X/F508del HNEs. * ( P ≤0.05) when compared with CFTR function in modulator treated F508del/Indel HNEs.

    Journal: PLoS Genetics

    Article Title: Capitalizing on the heterogeneous effects of CFTR nonsense and frameshift variants to inform therapeutic strategy for cystic fibrosis

    doi: 10.1371/journal.pgen.1007723

    Figure Lengend Snippet: CFTR nonsense variant with splicing defect has residual function that benefits from modulator treatments. (A) A schematic illustration of CFTR-Expression Minigene with introns 14–18 (introns 14 and 16 are full-length, and 15, 17, 18 are abridged). Arrow indicates location of E831X (top). CFTR mRNA splicing patterns of the total RNA extracted from HEK293 cells transiently transfected with E831X-EMG. (B) Steady state amounts of different isoforms of CFTR produced from E831X-EMG-i14-i18 expressed transiently in HEK293 cells. Lysates from cells expressing WT-EMG i14-i18, intronless WT CFTR or F508del served as positive controls, and empty vector as negative control. Immunoblot (IB) was probed with anti-CFTR antibody, 596 (CFFT). Horizontal arrows indicate to isoforms corresponding to (i) a normal codon (E831) substituted with a stop codon, (ii) deletion of complete exon 16, and (iii) deletion of a single amino acid E831. Beta-Actin was used as loading control. (C) Short-circuit (I sc ) tracing of CFTR function observed in CFBE-stable cells expressing E831X mounted on Ussing chamber. Cells were treated for 48 h with correctors (lumacaftor/tezacaftor or both, 3 μM each) and acutely with potentiator (ivacaftor, 10 μM). Change in I sc (ΔI sc ) was defined as the current inhibited by Inh-172 after sustained Isc responses achieved upon stimulation with forskolin alone or sequentially with ivacaftor. Data are presented as mean±SEM (n = 3). P value was determined by one way ANOVA. **** indicates significant difference ( P ≤0.0001) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated cells. WT-CFTR function represents forskolin stimulated Isc without modulator treatment in cells expressing EMG i14-i18. (D ) A tracing of CFTR function observed in primary nasal epithelial cells of an individual harboring E831X/F508del. CF-Human nasal epithelial (HNE) cells were treated for 24 h with lumacaftor and tezacaftor, 3 μM each, and acutely with Ivacaftor (10 μM). Stacked bar graph is a comparison of improvement in CFTR function of E831X/F508del vs F508del/Indel. Alternate Indel alleles were either 2184insA, 2183delAA > G, or 3659del C. P value was determined by one way ANOVA. ** indicates significant difference ( P ≤0.01) when compared with forskolin stimulated CFTR function in DMSO (vehicle) treated E831X/F508del HNEs. * ( P ≤0.05) when compared with CFTR function in modulator treated F508del/Indel HNEs.

    Article Snippet: After stabilization of transepithelial current, 10 μM forskolin (Selleckchem) was added to the basolateral chamber to stimulate generation of cAMP and activation of CFTR, followed by administration of 10 μM CFTR inhibitor-172 (Selleckchem) in the apical chamber to block CFTR-mediated currents.

    Techniques: Variant Assay, Expressing, Transfection, Produced, Plasmid Preparation, Negative Control

    Spatio-temporal cAMP dynamics in the sperm flagellum. ( A ) cAMP dynamics was analyzed in a region 20 µm in length in the midpiece (blue) and principal piece (green) of freely beating sperm. The cytoplasmic droplet is indicated with an arrow. ( B ) Changes in FRET after stimulation with 40 μM NKH477. The perfusion with NKH477 is indicated with a grey box. Data for a representative cell are shown as mean ± S.D. in the midpiece (blue) and principal piece (green). ( C ) Changes in FRET after stimulation with 25 mM bicarbonate. The perfusion with bicarbonate is indicated with a blue box. Individual traces have been fitted using logistic regression (Origin 9) (red line). ( D ) Average of the fitted data presented in ( C ). The blue and green line represent the mean value for the midpiece and principal piece, respectively (n = 7). The blue and green areas represent the corresponding S.D. DOI: http://dx.doi.org/10.7554/eLife.14052.013

    Journal: eLife

    Article Title: A novel biosensor to study cAMP dynamics in cilia and flagella

    doi: 10.7554/eLife.14052

    Figure Lengend Snippet: Spatio-temporal cAMP dynamics in the sperm flagellum. ( A ) cAMP dynamics was analyzed in a region 20 µm in length in the midpiece (blue) and principal piece (green) of freely beating sperm. The cytoplasmic droplet is indicated with an arrow. ( B ) Changes in FRET after stimulation with 40 μM NKH477. The perfusion with NKH477 is indicated with a grey box. Data for a representative cell are shown as mean ± S.D. in the midpiece (blue) and principal piece (green). ( C ) Changes in FRET after stimulation with 25 mM bicarbonate. The perfusion with bicarbonate is indicated with a blue box. Individual traces have been fitted using logistic regression (Origin 9) (red line). ( D ) Average of the fitted data presented in ( C ). The blue and green line represent the mean value for the midpiece and principal piece, respectively (n = 7). The blue and green areas represent the corresponding S.D. DOI: http://dx.doi.org/10.7554/eLife.14052.013

    Article Snippet: A simple "negative" control is perhaps to repeat the experiment in the presence of NKH477.

    Techniques:

    Characterisation of mlCNBD-FRET in HEK293 cells. ( A ) Calibration of mlCNBD-FRET in HEK293 cells. FRET was measured in a cuvette in a spectrofluorometer under basal conditions (ES), after permeabilization with 20 μM digitonin, and the following addition of increasing cAMP concentrations (in nM). According to the null-point calibration method, the difference in FRET ratio of the treated samples to the basal condition was determined and used to determine the basal cAMP concentration. ( B ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with 40 μM NKH477/500 μM IBMX (black). FRET has been measured using spectrofluorometer. Data are presented as mean ± S.D. DMSO (0.13%, red) has been used as a control; n = 3 for each condition. ( C ) Similar to part ( B ) after stimulation with 2 μM isoproterenol (black). ( D ) Similar to part ( B ) for stimulation with 3 mM SNP (black). ( E ) Change in the cerulean fluorescence lifetime measured using Fluorescence Lifetime Spectroscopy (FLS). Cells have been permeabilized with 20 μM digitonin followed by addition of cAMP. The cerulean fluorescence decay was recorded and fitted with a bi-exponential decay to calculate the lifetime. The mean of the two lifetimes was calculated and averaged over n = 3 experiments. ( F ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with different concentrations of NKH477. The FRET ratio has been calculated when reaching a maximum and normalized to the baseline ratio. Data is shown as mean ± S.D.; n = 4. ( G ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after alternatingly stimulating with 500 nM isoproterenol (black) followed by a wash-out with ES. As a control, cells were stimulated with buffer only (red). Data is shown as mean ± S.D.; n = 3. DOI: http://dx.doi.org/10.7554/eLife.14052.006

    Journal: eLife

    Article Title: A novel biosensor to study cAMP dynamics in cilia and flagella

    doi: 10.7554/eLife.14052

    Figure Lengend Snippet: Characterisation of mlCNBD-FRET in HEK293 cells. ( A ) Calibration of mlCNBD-FRET in HEK293 cells. FRET was measured in a cuvette in a spectrofluorometer under basal conditions (ES), after permeabilization with 20 μM digitonin, and the following addition of increasing cAMP concentrations (in nM). According to the null-point calibration method, the difference in FRET ratio of the treated samples to the basal condition was determined and used to determine the basal cAMP concentration. ( B ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with 40 μM NKH477/500 μM IBMX (black). FRET has been measured using spectrofluorometer. Data are presented as mean ± S.D. DMSO (0.13%, red) has been used as a control; n = 3 for each condition. ( C ) Similar to part ( B ) after stimulation with 2 μM isoproterenol (black). ( D ) Similar to part ( B ) for stimulation with 3 mM SNP (black). ( E ) Change in the cerulean fluorescence lifetime measured using Fluorescence Lifetime Spectroscopy (FLS). Cells have been permeabilized with 20 μM digitonin followed by addition of cAMP. The cerulean fluorescence decay was recorded and fitted with a bi-exponential decay to calculate the lifetime. The mean of the two lifetimes was calculated and averaged over n = 3 experiments. ( F ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with different concentrations of NKH477. The FRET ratio has been calculated when reaching a maximum and normalized to the baseline ratio. Data is shown as mean ± S.D.; n = 4. ( G ) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after alternatingly stimulating with 500 nM isoproterenol (black) followed by a wash-out with ES. As a control, cells were stimulated with buffer only (red). Data is shown as mean ± S.D.; n = 3. DOI: http://dx.doi.org/10.7554/eLife.14052.006

    Article Snippet: A simple "negative" control is perhaps to repeat the experiment in the presence of NKH477.

    Techniques: Concentration Assay, Expressing, Fluorescence, Spectroscopy

    Rapid generation of human GABAergic neurons by overexpression of Ascl1 and forskolin. ( a ) Culturing paradigm for the generation of induced GABAergic neurons (iGABAA-FSK). ( b ) iGABA A-FSK neuron immunostaining at DIV 49 for neuronal marker MAP2 co-labelled with Glutamate decarboxylase (GAD) 67 or GABA. ( c ) iGABA A-FSK are co-cultured from DIV 0 on with iGLU Ngn2 to promote functional maturation (named E/I networks), in a ratio of E/I 65:35. ( d ) VGAT and Gephyrin co-localisation in E/I networks at DIV 49. ( e ) Immunostaining for GABA co-labelled with either calbindin (CB), calretinin (CR), somatostatin (SST), parvalbumin (PV), MEF2C (asterix) or synaptotagmin-2 (SYT2, arrowheads) in E/I networks (Quantification sample size n= 7-9 coverslips per condition). ( f ) Heatmap showing expression of GABAergic/Glutamatergic transporters and – subtypes genes, and expression of genes important in GABAergic neuron development in E/I 65:35 networks at DIV 49 (3 biological replicates from one neuronal preparation). Data represents the log-transformed counts per million (logCPM). ( g ) Representative firing patterns of iGABA A+FSK neurons at DIV 28, −35 and −49. ( h-i ) Analysis of iGABA A+FSK membrane properties including ( h ) resting membrane potential (Vrmp) and ( i ) membrane capacitance (Cm). ( j-k ) Analysis of action potentials evoked by step-depolarization of iGABA A-FSK membranes including ( j ) fractions of maximum number of action potentials, and ( k ) Rheobase. ( l ) Quantifications of correlated synaptic input (number of synaptic burst/minute). ( m ) Spontaneous glutamatergic (red inset) and GABAergic (blue inset) postsynaptic inputs (sPSCs) received by iGLU Ngn2 . ( n ) Quantification of synaptic input types (Sample size for DIV 28 n=39, DIV 35 n=38, DIV 49 n=41 cells from 3 batches). All data represent means ± SEM. * p

    Journal: bioRxiv

    Article Title: Cadherin-13 is a critical regulator of GABAergic modulation in human stem cell derived neuronal networks

    doi: 10.1101/2020.05.07.082453

    Figure Lengend Snippet: Rapid generation of human GABAergic neurons by overexpression of Ascl1 and forskolin. ( a ) Culturing paradigm for the generation of induced GABAergic neurons (iGABAA-FSK). ( b ) iGABA A-FSK neuron immunostaining at DIV 49 for neuronal marker MAP2 co-labelled with Glutamate decarboxylase (GAD) 67 or GABA. ( c ) iGABA A-FSK are co-cultured from DIV 0 on with iGLU Ngn2 to promote functional maturation (named E/I networks), in a ratio of E/I 65:35. ( d ) VGAT and Gephyrin co-localisation in E/I networks at DIV 49. ( e ) Immunostaining for GABA co-labelled with either calbindin (CB), calretinin (CR), somatostatin (SST), parvalbumin (PV), MEF2C (asterix) or synaptotagmin-2 (SYT2, arrowheads) in E/I networks (Quantification sample size n= 7-9 coverslips per condition). ( f ) Heatmap showing expression of GABAergic/Glutamatergic transporters and – subtypes genes, and expression of genes important in GABAergic neuron development in E/I 65:35 networks at DIV 49 (3 biological replicates from one neuronal preparation). Data represents the log-transformed counts per million (logCPM). ( g ) Representative firing patterns of iGABA A+FSK neurons at DIV 28, −35 and −49. ( h-i ) Analysis of iGABA A+FSK membrane properties including ( h ) resting membrane potential (Vrmp) and ( i ) membrane capacitance (Cm). ( j-k ) Analysis of action potentials evoked by step-depolarization of iGABA A-FSK membranes including ( j ) fractions of maximum number of action potentials, and ( k ) Rheobase. ( l ) Quantifications of correlated synaptic input (number of synaptic burst/minute). ( m ) Spontaneous glutamatergic (red inset) and GABAergic (blue inset) postsynaptic inputs (sPSCs) received by iGLU Ngn2 . ( n ) Quantification of synaptic input types (Sample size for DIV 28 n=39, DIV 35 n=38, DIV 49 n=41 cells from 3 batches). All data represent means ± SEM. * p

    Article Snippet: At DIV 1 cultures were switched to DMEM/F12 containing Forskolin (10 μM, Sigma), N2 (1:100, Thermo Fisher Scientific), non-essential amino acids (1:100, Sigma), primocin (0.1 μg/ml), NT3 (10 ng/ml), BDNF (10 ng/ml), and doxycycline (4μg/ml).

    Techniques: Over Expression, Immunostaining, Marker, Cell Culture, Functional Assay, Expressing, Transformation Assay

    Blockade of ramelteon-induced potentiation of CREB phosphorylation by luzindole and forskolin. (A) INS-1 cells were incubated with ramelteon (5 nM) in the absence or presence of luzindole (15 µM) or forskolin (0.1 µM) for 14 h. After drug washout, the cells were subjected to a second round of forskolin stimulation (0.1 µM) for 30 min. (B) INS-1 cells were incubated with 2′,5′-dideoxyadenosine (10 or 100 µM) and H89 (1 or 10 µM) for 2 or 14 h. After drug washout, the cells were stimulated with forskolin (0.1 µM) for 30 min. Values are expressed as the ratio of phosphorylated CREB to total CREB in the vehicle-pretreated control (100%). Data are presented as means ± SEM (n = 3–6) and were analyzed using 2-way analysis of variance followed by Dunnett's test. *** P

    Journal: PLoS ONE

    Article Title: The Melatonin Agonist Ramelteon Induces Duration-Dependent Clock Gene Expression through cAMP Signaling in Pancreatic INS-1 ?-Cells

    doi: 10.1371/journal.pone.0102073

    Figure Lengend Snippet: Blockade of ramelteon-induced potentiation of CREB phosphorylation by luzindole and forskolin. (A) INS-1 cells were incubated with ramelteon (5 nM) in the absence or presence of luzindole (15 µM) or forskolin (0.1 µM) for 14 h. After drug washout, the cells were subjected to a second round of forskolin stimulation (0.1 µM) for 30 min. (B) INS-1 cells were incubated with 2′,5′-dideoxyadenosine (10 or 100 µM) and H89 (1 or 10 µM) for 2 or 14 h. After drug washout, the cells were stimulated with forskolin (0.1 µM) for 30 min. Values are expressed as the ratio of phosphorylated CREB to total CREB in the vehicle-pretreated control (100%). Data are presented as means ± SEM (n = 3–6) and were analyzed using 2-way analysis of variance followed by Dunnett's test. *** P

    Article Snippet: Forskolin was procured from Merck Millipore (Darmstadt, Germany).

    Techniques: Incubation

    Duration-dependent changes in ramelteon-induced clock gene expression. INS-1 cells were treated with ramelteon (A and C; 10 nM) and forskolin (B and D; 0.1 µM) for 2, 4, 8, and 14 h. mRNA expression of Rev-erbα , Bmal1 , Clock , Per1 , Per2 , Cry1 , and Cry2 was assessed using TaqMan polymerase chain reaction and normalized to that of the housekeeping gene cyclophilin A. Values are expressed as a percentage of the vehicle-treated control at each time point. Data are presented as means ± SEM (n = 4).

    Journal: PLoS ONE

    Article Title: The Melatonin Agonist Ramelteon Induces Duration-Dependent Clock Gene Expression through cAMP Signaling in Pancreatic INS-1 ?-Cells

    doi: 10.1371/journal.pone.0102073

    Figure Lengend Snippet: Duration-dependent changes in ramelteon-induced clock gene expression. INS-1 cells were treated with ramelteon (A and C; 10 nM) and forskolin (B and D; 0.1 µM) for 2, 4, 8, and 14 h. mRNA expression of Rev-erbα , Bmal1 , Clock , Per1 , Per2 , Cry1 , and Cry2 was assessed using TaqMan polymerase chain reaction and normalized to that of the housekeeping gene cyclophilin A. Values are expressed as a percentage of the vehicle-treated control at each time point. Data are presented as means ± SEM (n = 4).

    Article Snippet: Forskolin was procured from Merck Millipore (Darmstadt, Germany).

    Techniques: Expressing, Polymerase Chain Reaction

    Duration-dependent changes in insulin secretion during ramelteon treatment and after drug washout. (A) INS-1 cells were treated with ramelteon (0.1–100 nM) for 2 or 14 h. (B) After ramelteon treatment for 2 or 14 h, the cells were washed twice and stimulated with forskolin (10 µM) for 2 h in the absence of ramelteon. (C) The cells were incubated with ramelteon (10 nM) in the absence or presence of luzindole (30 µM) for 14 h. After drug washout, the cells were stimulated with forskolin (10 µM) for 2 h. Values are expressed as the percentage of the vehicle- (A) or forskolin (10 µM; B and C)-stimulated insulin release in vehicle-pretreated controls. Data are presented as means ± SEM (n = 6) and were analyzed using 2-way analysis of variance followed by Dunnett's test. *** P

    Journal: PLoS ONE

    Article Title: The Melatonin Agonist Ramelteon Induces Duration-Dependent Clock Gene Expression through cAMP Signaling in Pancreatic INS-1 ?-Cells

    doi: 10.1371/journal.pone.0102073

    Figure Lengend Snippet: Duration-dependent changes in insulin secretion during ramelteon treatment and after drug washout. (A) INS-1 cells were treated with ramelteon (0.1–100 nM) for 2 or 14 h. (B) After ramelteon treatment for 2 or 14 h, the cells were washed twice and stimulated with forskolin (10 µM) for 2 h in the absence of ramelteon. (C) The cells were incubated with ramelteon (10 nM) in the absence or presence of luzindole (30 µM) for 14 h. After drug washout, the cells were stimulated with forskolin (10 µM) for 2 h. Values are expressed as the percentage of the vehicle- (A) or forskolin (10 µM; B and C)-stimulated insulin release in vehicle-pretreated controls. Data are presented as means ± SEM (n = 6) and were analyzed using 2-way analysis of variance followed by Dunnett's test. *** P

    Article Snippet: Forskolin was procured from Merck Millipore (Darmstadt, Germany).

    Techniques: Incubation

    Duration-dependent changes in CREB phosphorylation during ramelteon treatment and after washout. (A) INS-1 cells were treated with ramelteon (10 nM) for 2, 4, 8, or 14 h. (B) Concentration-dependent decreases in CREB phosphorylation were assessed after ramelteon (0.1–100 nM) treatment for 2 and 14 h. (C) After ramelteon (10 nM) treatment for 2, 4, 8, or 14 h, the cells were washed twice and stimulated with forskolin (0.1 µM) for 30 min in the absence of ramelteon. (D) Concentration-dependent potentiation of forskolin-stimulated CREB phosphorylation was assessed after ramelteon (0.1–100 nM) treatment for 2 and 14 h. Values are expressed as the ratio of phosphorylated CREB to total CREB in the vehicle-pretreated control (100%). Data are presented as means ± SEM (n = 3) and were analyzed using 2-way analysis of variance followed by Dunnett's test. *** P

    Journal: PLoS ONE

    Article Title: The Melatonin Agonist Ramelteon Induces Duration-Dependent Clock Gene Expression through cAMP Signaling in Pancreatic INS-1 ?-Cells

    doi: 10.1371/journal.pone.0102073

    Figure Lengend Snippet: Duration-dependent changes in CREB phosphorylation during ramelteon treatment and after washout. (A) INS-1 cells were treated with ramelteon (10 nM) for 2, 4, 8, or 14 h. (B) Concentration-dependent decreases in CREB phosphorylation were assessed after ramelteon (0.1–100 nM) treatment for 2 and 14 h. (C) After ramelteon (10 nM) treatment for 2, 4, 8, or 14 h, the cells were washed twice and stimulated with forskolin (0.1 µM) for 30 min in the absence of ramelteon. (D) Concentration-dependent potentiation of forskolin-stimulated CREB phosphorylation was assessed after ramelteon (0.1–100 nM) treatment for 2 and 14 h. Values are expressed as the ratio of phosphorylated CREB to total CREB in the vehicle-pretreated control (100%). Data are presented as means ± SEM (n = 3) and were analyzed using 2-way analysis of variance followed by Dunnett's test. *** P

    Article Snippet: Forskolin was procured from Merck Millipore (Darmstadt, Germany).

    Techniques: Concentration Assay

    Blockade of ramelteon-induced Rev-erbα expression by luzindole and forskolin. INS-1 cells were incubated with ramelteon (A and B, 5 nM; C and D, 10 nM) in the absence or presence of luzindole (A and B, 15 µM) or forskolin (C and D, 0.1 µM) for 14 h. mRNA expression of Rev-erbα and Bmal1 was measured using TaqMan polymerase chain reaction and normalized to that of the housekeeping gene cyclophilin A. Values are expressed as a percentage of vehicle-treated controls. Data are presented as means ± SEM (n = 4−6) and were analyzed using 2-way analysis of variance followed by Dunnett's test. ** P

    Journal: PLoS ONE

    Article Title: The Melatonin Agonist Ramelteon Induces Duration-Dependent Clock Gene Expression through cAMP Signaling in Pancreatic INS-1 ?-Cells

    doi: 10.1371/journal.pone.0102073

    Figure Lengend Snippet: Blockade of ramelteon-induced Rev-erbα expression by luzindole and forskolin. INS-1 cells were incubated with ramelteon (A and B, 5 nM; C and D, 10 nM) in the absence or presence of luzindole (A and B, 15 µM) or forskolin (C and D, 0.1 µM) for 14 h. mRNA expression of Rev-erbα and Bmal1 was measured using TaqMan polymerase chain reaction and normalized to that of the housekeeping gene cyclophilin A. Values are expressed as a percentage of vehicle-treated controls. Data are presented as means ± SEM (n = 4−6) and were analyzed using 2-way analysis of variance followed by Dunnett's test. ** P

    Article Snippet: Forskolin was procured from Merck Millipore (Darmstadt, Germany).

    Techniques: Expressing, Incubation, Polymerase Chain Reaction

    Time course of clock gene expression after ramelteon pretreatment followed by forskolin and high glucose stimulation. INS-1 cells were pretreated with ramelteon (10 nM) for 2 h or 14 h (A–D). After the washout period, the cells were stimulated with forskolin (0.1 µM) and high glucose (12 mM) for 1.5 h. Following removal of the stimulant, the cells were incubated in a serum-free medium for 0–48 h. Clock gene expression was assessed by TaqMan polymerase chain reaction and normalized to that of the housekeeping gene cyclophilin A. Values are expressed as the percentage of vehicle-stimulated controls at 0 h after stimulant removal. Data are presented as means ± SEM (n = 3) and were analyzed using 2-way analysis of variance followed by Dunnett's test. # P

    Journal: PLoS ONE

    Article Title: The Melatonin Agonist Ramelteon Induces Duration-Dependent Clock Gene Expression through cAMP Signaling in Pancreatic INS-1 ?-Cells

    doi: 10.1371/journal.pone.0102073

    Figure Lengend Snippet: Time course of clock gene expression after ramelteon pretreatment followed by forskolin and high glucose stimulation. INS-1 cells were pretreated with ramelteon (10 nM) for 2 h or 14 h (A–D). After the washout period, the cells were stimulated with forskolin (0.1 µM) and high glucose (12 mM) for 1.5 h. Following removal of the stimulant, the cells were incubated in a serum-free medium for 0–48 h. Clock gene expression was assessed by TaqMan polymerase chain reaction and normalized to that of the housekeeping gene cyclophilin A. Values are expressed as the percentage of vehicle-stimulated controls at 0 h after stimulant removal. Data are presented as means ± SEM (n = 3) and were analyzed using 2-way analysis of variance followed by Dunnett's test. # P

    Article Snippet: Forskolin was procured from Merck Millipore (Darmstadt, Germany).

    Techniques: Expressing, Incubation, Polymerase Chain Reaction

    PKA acts downstream R-Smads to mediate anti-motility signaling of TGFβ3. ( A ) HDFs were infected with FG-12 lentivirus carrying either a GFP control gene or a shRNAs against PKA-Cα. After 48 hours, downregulation of the endogenous PKA-Cα was confirmed by immunoblotting the lysates of the cells with an anti-PKA-Cα antibody. ( B ) Downregulation of PKA-Cα blocks forskolin-stimulated CREB phosphorylation (lane 4 versus lane 2). ( C ) Downregulation of PKA-Cα showed little effect on TGFβ3-stimulated phosphorylation of Smad3 (lanes 4 and 6 versus lane 2). ( D ) Two sets of PKA-Cα-downregulated HDFs were subjected to colloidal gold migration assay in response to PDGF-bb (15 ng/ml) in the absence or presence of TGFβ3 (3 ng/ml). Representative images of the migrated cells under the indicated conditions are shown (a to i). ( E ) The computer-assisted quantitative analyses of the migration tracks are shown as Migration index (MI) (%). The experiment was repeated four times ( n = 3, P ≤0.05). *Statistically significant over the control.

    Journal: Biology Open

    Article Title: The anti-motility signaling mechanism of TGF?3 that controls cell traffic during skin wound healing

    doi: 10.1242/bio.20122246

    Figure Lengend Snippet: PKA acts downstream R-Smads to mediate anti-motility signaling of TGFβ3. ( A ) HDFs were infected with FG-12 lentivirus carrying either a GFP control gene or a shRNAs against PKA-Cα. After 48 hours, downregulation of the endogenous PKA-Cα was confirmed by immunoblotting the lysates of the cells with an anti-PKA-Cα antibody. ( B ) Downregulation of PKA-Cα blocks forskolin-stimulated CREB phosphorylation (lane 4 versus lane 2). ( C ) Downregulation of PKA-Cα showed little effect on TGFβ3-stimulated phosphorylation of Smad3 (lanes 4 and 6 versus lane 2). ( D ) Two sets of PKA-Cα-downregulated HDFs were subjected to colloidal gold migration assay in response to PDGF-bb (15 ng/ml) in the absence or presence of TGFβ3 (3 ng/ml). Representative images of the migrated cells under the indicated conditions are shown (a to i). ( E ) The computer-assisted quantitative analyses of the migration tracks are shown as Migration index (MI) (%). The experiment was repeated four times ( n = 3, P ≤0.05). *Statistically significant over the control.

    Article Snippet: Anti-PKA Cα antibody, H-89 (dihydrochloride) and forskolin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma–Aldrich (St. Louis, MO), respectively.

    Techniques: Infection, Migration

    Activation of PKA alone replaces the anti-motility signal of TGFβ3 in HDFs. ( A ) HDFs were serum starved overnight and subjected to colloidal gold migration assays in the absence or presence of PDGF-bb (15 ng/ml), TGFβ3 (3 ng/ml) and PKA inhibitor, H89 (1.0 µM). Representative images of migrated cells under the indicated conditions are shown (a to d). Quantitative analyses of the migration tracks are shown underneath as Migration index (MI) (%). Average size migration tracks are marked with dotted circles. This experiment was repeated multiple times and similar results were obtained. ( B ) Serum-starved HDFs were treated without or with TGFβ3 or TGFβ3 plus H89. Total cell lysates were subjected to immunoblotting analyses with indicated antibodies. ( C ) Serum-starved HDFs were subjected to colloidal gold migration assay, as previously described, in the absence or presence of PDGF-bb (bar 1 and 2) or PDGF-bb plus increasing concentrations of forskolin, an activator of PKA (bars 3 to 7). The effectiveness of forskolin (10.0 µM) on HDFs is indicated by causing an increased phosphorylation of CREB (insert image) in the cells. These experiments were repeated three times. *Statistically significant over the control, P

    Journal: Biology Open

    Article Title: The anti-motility signaling mechanism of TGF?3 that controls cell traffic during skin wound healing

    doi: 10.1242/bio.20122246

    Figure Lengend Snippet: Activation of PKA alone replaces the anti-motility signal of TGFβ3 in HDFs. ( A ) HDFs were serum starved overnight and subjected to colloidal gold migration assays in the absence or presence of PDGF-bb (15 ng/ml), TGFβ3 (3 ng/ml) and PKA inhibitor, H89 (1.0 µM). Representative images of migrated cells under the indicated conditions are shown (a to d). Quantitative analyses of the migration tracks are shown underneath as Migration index (MI) (%). Average size migration tracks are marked with dotted circles. This experiment was repeated multiple times and similar results were obtained. ( B ) Serum-starved HDFs were treated without or with TGFβ3 or TGFβ3 plus H89. Total cell lysates were subjected to immunoblotting analyses with indicated antibodies. ( C ) Serum-starved HDFs were subjected to colloidal gold migration assay, as previously described, in the absence or presence of PDGF-bb (bar 1 and 2) or PDGF-bb plus increasing concentrations of forskolin, an activator of PKA (bars 3 to 7). The effectiveness of forskolin (10.0 µM) on HDFs is indicated by causing an increased phosphorylation of CREB (insert image) in the cells. These experiments were repeated three times. *Statistically significant over the control, P

    Article Snippet: Anti-PKA Cα antibody, H-89 (dihydrochloride) and forskolin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma–Aldrich (St. Louis, MO), respectively.

    Techniques: Activation Assay, Migration

    I SC and C T recordings with and without hormonal supplementation. (A) Representative I SC (black trace) and C T (grey trace) recording from mpkCCD cells mounted in modified Ussing chambers and stimulated with (10 µM) forskolin. Addition of 10 µM amiloride at the end of the trace demonstrated the majority of the recorded I SC was Na + transport via ENaC. (B) A similar trace from mpkCCD cells cultured in the absence of dexamethasone supplementation. (C) Summarized data for stimulated amiloride-sensitive current (I Na ) and C T response to forskolin stimulation (n = 14) in cells with (+) and without (-) full supplementation.

    Journal: PLoS ONE

    Article Title: The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

    doi: 10.1371/journal.pone.0046593

    Figure Lengend Snippet: I SC and C T recordings with and without hormonal supplementation. (A) Representative I SC (black trace) and C T (grey trace) recording from mpkCCD cells mounted in modified Ussing chambers and stimulated with (10 µM) forskolin. Addition of 10 µM amiloride at the end of the trace demonstrated the majority of the recorded I SC was Na + transport via ENaC. (B) A similar trace from mpkCCD cells cultured in the absence of dexamethasone supplementation. (C) Summarized data for stimulated amiloride-sensitive current (I Na ) and C T response to forskolin stimulation (n = 14) in cells with (+) and without (-) full supplementation.

    Article Snippet: A typical cAMP stimulation involved the addition of 10 µM forskolin (Fisher Scientific, Pittsburgh, PA) basolaterally, which produced a maximum ISC stimulation after 10–20 min. To determine the net Na+ transport through ENaC, 10 µM amiloride (Sigma) was added to the apical cell surface at the end of each experiment.

    Techniques: Modification, Cell Culture

    Confocal fluorescent images of FM1-43X endocytosis in mpkCCD cells. (A) Representative maximum projection images from confocal stacks obtained at 0 min and 10 min in basal (no forskolin) and forskolin stimulated (10 µM) cells previously cultured in fully supplemented media. (B) Representative images as in (A) from cells cultured without dexamethasone supplementation. (Bars represent 10 µm). (C) The number of vesicles internalized per cell is presented as a percentage of the fully supplemented counts at time 0 min (N = 3, n > 200). Counts from cells cultured without dexamethasone supplementation (Unsupplemented) were significantly lower than cells in full supplementation for all conditions (p

    Journal: PLoS ONE

    Article Title: The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

    doi: 10.1371/journal.pone.0046593

    Figure Lengend Snippet: Confocal fluorescent images of FM1-43X endocytosis in mpkCCD cells. (A) Representative maximum projection images from confocal stacks obtained at 0 min and 10 min in basal (no forskolin) and forskolin stimulated (10 µM) cells previously cultured in fully supplemented media. (B) Representative images as in (A) from cells cultured without dexamethasone supplementation. (Bars represent 10 µm). (C) The number of vesicles internalized per cell is presented as a percentage of the fully supplemented counts at time 0 min (N = 3, n > 200). Counts from cells cultured without dexamethasone supplementation (Unsupplemented) were significantly lower than cells in full supplementation for all conditions (p

    Article Snippet: A typical cAMP stimulation involved the addition of 10 µM forskolin (Fisher Scientific, Pittsburgh, PA) basolaterally, which produced a maximum ISC stimulation after 10–20 min. To determine the net Na+ transport through ENaC, 10 µM amiloride (Sigma) was added to the apical cell surface at the end of each experiment.

    Techniques: Cell Culture

    ENaC siRNA reduces I SC and cAMP C T response. (A) Western blots of whole cell lysate from βENaC knockdown demonstrate reduction in expression of ENaC compared to control siRNA transfected mpkCCD cells. The actin-corrected percent reduction in expression (n = 3) are summarized to the right of each representative blot. (B) Representative traces for I SC (top traces) and C T recordings (bottom) for control (black traces) and ENaC knockdown (grey traces) cells stimulated with forskolin (10 µM). (C) Summarized data for I SC versus C T responses to forskolin stimulation for control (n = 45) and βENaC (n = 55) knockdown cells similar to those presented in (B).

    Journal: PLoS ONE

    Article Title: The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

    doi: 10.1371/journal.pone.0046593

    Figure Lengend Snippet: ENaC siRNA reduces I SC and cAMP C T response. (A) Western blots of whole cell lysate from βENaC knockdown demonstrate reduction in expression of ENaC compared to control siRNA transfected mpkCCD cells. The actin-corrected percent reduction in expression (n = 3) are summarized to the right of each representative blot. (B) Representative traces for I SC (top traces) and C T recordings (bottom) for control (black traces) and ENaC knockdown (grey traces) cells stimulated with forskolin (10 µM). (C) Summarized data for I SC versus C T responses to forskolin stimulation for control (n = 45) and βENaC (n = 55) knockdown cells similar to those presented in (B).

    Article Snippet: A typical cAMP stimulation involved the addition of 10 µM forskolin (Fisher Scientific, Pittsburgh, PA) basolaterally, which produced a maximum ISC stimulation after 10–20 min. To determine the net Na+ transport through ENaC, 10 µM amiloride (Sigma) was added to the apical cell surface at the end of each experiment.

    Techniques: Western Blot, Expressing, Transfection

    ENaC expression in FRT cells . (A) Representative I SC trace from FTR epithelial cells transfected with α,β,γENaC (black trace) or control plasmid (grey trace), mounted in Ussing chambers and stimulated with 10 µM forskolin (grey bar). The expression of ENaC was confirmed by the addition of 10 µM amiloride (black bar) at the end of the recording. (B) Data from a number of similar experiments (n = 34) are summarized. Basal (-cAMP) and forskolin-stimulated (+cAMP) I Na are presented on the left and the change in I SC with foskolin stimulation (ΔI SC ) on the right (bar graph). (C) Representative capacitance trace from the same samples provided in (A). (D) Summarized data for capacitance changes under basal and forskolin-stimulated conditions as in (B). (* - indicates significantly different from untransfected p

    Journal: PLoS ONE

    Article Title: The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

    doi: 10.1371/journal.pone.0046593

    Figure Lengend Snippet: ENaC expression in FRT cells . (A) Representative I SC trace from FTR epithelial cells transfected with α,β,γENaC (black trace) or control plasmid (grey trace), mounted in Ussing chambers and stimulated with 10 µM forskolin (grey bar). The expression of ENaC was confirmed by the addition of 10 µM amiloride (black bar) at the end of the recording. (B) Data from a number of similar experiments (n = 34) are summarized. Basal (-cAMP) and forskolin-stimulated (+cAMP) I Na are presented on the left and the change in I SC with foskolin stimulation (ΔI SC ) on the right (bar graph). (C) Representative capacitance trace from the same samples provided in (A). (D) Summarized data for capacitance changes under basal and forskolin-stimulated conditions as in (B). (* - indicates significantly different from untransfected p

    Article Snippet: A typical cAMP stimulation involved the addition of 10 µM forskolin (Fisher Scientific, Pittsburgh, PA) basolaterally, which produced a maximum ISC stimulation after 10–20 min. To determine the net Na+ transport through ENaC, 10 µM amiloride (Sigma) was added to the apical cell surface at the end of each experiment.

    Techniques: Expressing, Transfection, Plasmid Preparation

    Aldosterone restores I SC and C T responses to cAMP stimulation. (A) Cells in unsupplemented media were incubated with 100 nM aldosterone for increasing time and stimulated with forskolin. The basal (-cAMP) and forskolin stimulated (+cAMP) currents from cells with no aldosterone (unstimulated), 6, 12 and 24 hr aldosterone stimulation were compared to cells which received fully supplemented medium (supplemented). The average I SC response to cAMP stimulation is plotted as a bar graph on the right (n = 16). (B) The change in capacitance (ΔC T ) versus change in amiloride-sensitive I SC (ΔI Na ) in response to forskolin stimulation is summarized for cells without supplementation and 24 hr stimulation with aldosterone. Both I SC and C T responses were significantly greater in the aldosterone treated cells compared to unsupplemented controls (n = 16, p

    Journal: PLoS ONE

    Article Title: The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

    doi: 10.1371/journal.pone.0046593

    Figure Lengend Snippet: Aldosterone restores I SC and C T responses to cAMP stimulation. (A) Cells in unsupplemented media were incubated with 100 nM aldosterone for increasing time and stimulated with forskolin. The basal (-cAMP) and forskolin stimulated (+cAMP) currents from cells with no aldosterone (unstimulated), 6, 12 and 24 hr aldosterone stimulation were compared to cells which received fully supplemented medium (supplemented). The average I SC response to cAMP stimulation is plotted as a bar graph on the right (n = 16). (B) The change in capacitance (ΔC T ) versus change in amiloride-sensitive I SC (ΔI Na ) in response to forskolin stimulation is summarized for cells without supplementation and 24 hr stimulation with aldosterone. Both I SC and C T responses were significantly greater in the aldosterone treated cells compared to unsupplemented controls (n = 16, p

    Article Snippet: A typical cAMP stimulation involved the addition of 10 µM forskolin (Fisher Scientific, Pittsburgh, PA) basolaterally, which produced a maximum ISC stimulation after 10–20 min. To determine the net Na+ transport through ENaC, 10 µM amiloride (Sigma) was added to the apical cell surface at the end of each experiment.

    Techniques: Incubation

    Inhibiting ENaC activity does not alter trafficking. (A) Representative I SC (black trace) and C T (grey trace) traces from mpkCCD cells pretreated with 50 µM FCI for 24 hrs before recordings and stimulated with forskolin (10 µM). (B) Single I SC recording from FCI inhibited cells (as in A) where 1 µM trypsin was added to the apical bath (light blue bar) to activate uncleaved channels. (C) Summarized I SC versus ΔC T plots for control unsupplemented, aldosterone treated and aldo+FCI treated cells (n > 16 per point). The ΔC T for uncleaved channels was significantly greater (p

    Journal: PLoS ONE

    Article Title: The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

    doi: 10.1371/journal.pone.0046593

    Figure Lengend Snippet: Inhibiting ENaC activity does not alter trafficking. (A) Representative I SC (black trace) and C T (grey trace) traces from mpkCCD cells pretreated with 50 µM FCI for 24 hrs before recordings and stimulated with forskolin (10 µM). (B) Single I SC recording from FCI inhibited cells (as in A) where 1 µM trypsin was added to the apical bath (light blue bar) to activate uncleaved channels. (C) Summarized I SC versus ΔC T plots for control unsupplemented, aldosterone treated and aldo+FCI treated cells (n > 16 per point). The ΔC T for uncleaved channels was significantly greater (p

    Article Snippet: A typical cAMP stimulation involved the addition of 10 µM forskolin (Fisher Scientific, Pittsburgh, PA) basolaterally, which produced a maximum ISC stimulation after 10–20 min. To determine the net Na+ transport through ENaC, 10 µM amiloride (Sigma) was added to the apical cell surface at the end of each experiment.

    Techniques: Activity Assay

    Involvement of PKC isoforms in EPAC-dependent SOCS-3 induction in COS1 cells. a , COS1 cells were stimulated for 5 h with MG132 (10 μ m ) plus 50 μ m 8Me, a combination of 10 μ m forskolin plus 10 μ m rolipram (F/R) or 10 μ

    Journal: The Journal of Biological Chemistry

    Article Title: Activation of Protein Kinase C? by EPAC1 Is Required for the ERK- and CCAAT/Enhancer-binding Protein ?-dependent Induction of the SOCS-3 Gene by Cyclic AMP in COS1 Cells *

    doi: 10.1074/jbc.M109.015370

    Figure Lengend Snippet: Involvement of PKC isoforms in EPAC-dependent SOCS-3 induction in COS1 cells. a , COS1 cells were stimulated for 5 h with MG132 (10 μ m ) plus 50 μ m 8Me, a combination of 10 μ m forskolin plus 10 μ m rolipram (F/R) or 10 μ

    Article Snippet: Phorbol 12-myristate 13-acetate, BAPTA-AM, Ro-31-7549, GF 109203X, , , U0126, MG132, forskolin, and rolipram were purchased from Merck.

    Techniques:

    Elevation of cyclic AMP promotes EPAC-dependent activation of PKCα in COS1 cells . a , COS1 cells were stimulated for the indicated times with either 10 μ m forskolin plus 10 μ m rolipram ( F/R ) or 10 μ m PMA. Cells were then

    Journal: The Journal of Biological Chemistry

    Article Title: Activation of Protein Kinase C? by EPAC1 Is Required for the ERK- and CCAAT/Enhancer-binding Protein ?-dependent Induction of the SOCS-3 Gene by Cyclic AMP in COS1 Cells *

    doi: 10.1074/jbc.M109.015370

    Figure Lengend Snippet: Elevation of cyclic AMP promotes EPAC-dependent activation of PKCα in COS1 cells . a , COS1 cells were stimulated for the indicated times with either 10 μ m forskolin plus 10 μ m rolipram ( F/R ) or 10 μ m PMA. Cells were then

    Article Snippet: Phorbol 12-myristate 13-acetate, BAPTA-AM, Ro-31-7549, GF 109203X, , , U0126, MG132, forskolin, and rolipram were purchased from Merck.

    Techniques: Activation Assay

    Involvement of PLCϵ in SOCS-3 induction by cyclic AMP in COS1 cells . a , COS1 cells were stimulated for 5 h with the proteosome inhibitor MG132 (10 μ m ) and 10 μ m forskolin plus 10 μ m rolipram ( F/R ) in the presence or absence

    Journal: The Journal of Biological Chemistry

    Article Title: Activation of Protein Kinase C? by EPAC1 Is Required for the ERK- and CCAAT/Enhancer-binding Protein ?-dependent Induction of the SOCS-3 Gene by Cyclic AMP in COS1 Cells *

    doi: 10.1074/jbc.M109.015370

    Figure Lengend Snippet: Involvement of PLCϵ in SOCS-3 induction by cyclic AMP in COS1 cells . a , COS1 cells were stimulated for 5 h with the proteosome inhibitor MG132 (10 μ m ) and 10 μ m forskolin plus 10 μ m rolipram ( F/R ) in the presence or absence

    Article Snippet: Phorbol 12-myristate 13-acetate, BAPTA-AM, Ro-31-7549, GF 109203X, , , U0126, MG132, forskolin, and rolipram were purchased from Merck.

    Techniques: