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  • 99
    Millipore cell permeable vivit
    Impairing calcineurin/NFAT signaling results in hair growth and stem cell proliferation (A) Schematic illustrating the experimental design of CSA experiments. (B-C) Quantification of anagen induction and BrdU incorporation in follicle stem cells of the bulge (Bu) with <t>cyclosporine</t> A (CSA) treatment. The anagen control used was the first hair cycle (P18-P25). Data are the mean ± SEM for 50−100 follicles for 3 individual mice for each timepoint. Asterisks in (C) indicate hair shaft autofluorescence. (D-E) Immunohistochemistry and western analysis of NFATc1 expression following 3d treatment with vehicle or CSA. (F) Colony formation and cell number of bulge cells following treatment with vehicle, CSA, <t>11R-VIVIT,</t> or 11R-VEET. N= 3 experiments with independent sorted populations. (G) NFAT reporter activity in response to calcium ionophore (I), phorbol myristate acetate (PMA), caNFATc1, CSA or 11R-VIVIT. Data are mean ± SEM. N=3−5 individual experiments for each treatment. Asterisks indicate significance, p
    Cell Permeable Vivit, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore vivit
    Sildenafil inhibits hypoxia-induced nuclear translocation of NFATc3 in cultured human PASMC . Human PASMC were cultured with SMBM (2% FBS) under nomoxia or hypoxia (3% O 2 ) in the presence of sildenafil (100 nM), <t>8-brom-cGMP</t> (100 μM), SKF96365 (7.5 μM) or <t>VIVIT</t> (4 μM) respectively for 72 h. NFATc3 was determined by confocal microscopy of immunofluorescence. The primary antibody of NFATc3 was detected with Rhodamine (TRITC)-conjugated AffiniPure Goat Anti-mouse IgG (green). Slides were counterstained with nuclei dye hoechest33258 (blue). A: Immunofluorescence image of NFATc3 in human PASMC (×1000). B: The nuclear translocation of NFATc3 was calculated by comparing the ratio of nuclear NFATc3 immunofluorescence/total NFATc3 immunofluorescence. n = 20, ### P
    Vivit, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris vivit
    Regulation of IL2 by NFAT inferred by dynamic Boolean model: (a) network of interactions between IL2 and the TFs regulating its expression assembled from literature, (b) enhanced regulatory network inferred using dynamic Boolean model, and (c) the expression of IL2 mRNA evaluated by RT-PCR in <t>DCs</t> treated for one hour with medium containing the NFAT inhibitors CsA (CsA), <t>VIVIT</t> peptide (VIVIT), or vehicle (−), and then left mock-infected (mock) or infected with IV during 2 hours. The expression levels of IL2 in infected cells were normalized to the levels of nontreated, mock-infected cells.
    Vivit, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Merck KGaA 11r vivit
    Effects of <t>11R-VIVIT</t> and NFAT2 gene knockdown on the expression of the uPA receptor (Plaur) gene in podocytes treated with high glucose. (A)The mRNA level of uPA receptors was examined using real-time quantitative RT-PCR analysis, and GAPDH mRNA was used as an internal control. Quantitative data were calculated by 2 -△△ CT . (B) The protein level of uPA receptors was analysed using Western blot analysis( n = 3). (C) Densitometry analysis of three repetitions of the experiment shown in (B). Note NG, normal glucose (5.3 mM) group; HG, high glucose group(30 mM); MA, normal glucose (5.3 mM) + mannitol(24.7 mM)group, as an osmolality control; HG+11R-VIVIT, High glucose (30 mM)+ 11R-VIVIT(100 nM, a cell permeable NFAT inhibitor) group; HG + NFAT2-siRNA, High glucose (30 mM) + NFAT2-siRNA (50 nM) group; HG + control-siRNA, High glucose (30 mM) + control-siRNA (50 nM), as a negative control group. All Values are expressed as the mean ± SEM. * P
    11r Vivit, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 89/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc vivit
    Effect of v-FLIP/K13 on COX-2, mPGES-1 gene expression, COX-2 and COX-1 protein levels and PGE2 secretion and role of cis -acting factors on transcriptional regulation of COX-2 promoter by v-FLIP/K13. ( a , b ) HMVEC-d cells were infected with KSHV latency gene (LANA-1, v-FLIP, v-Cyclin, K8 and K12), GFP or empty vector (pSIN) expressing lentiviruses. Forty-eight hours after infection, cells were washed, lysed, total RNA was prepared, and COX-2 and mPGES-1 expression were quantitated using COX-2 and mPGES-1 gene-specific primers normalized to GAPDH levels as described earlier. 5 Fold induction was calculated by considering levels in pSIN lentivirus-infected cells as onefold. ( c ) Supernatants collected from cells in ( a ) were used for PGE2 detection by methods described previously. 5 Each reaction was done in triplicate and each point represents the average±s.d. of three independent experiments. ( d ) Lysates prepared from the above mentioned cells were used for quantitating COX-2 protein levels. ( e ) A total of 293 cells seeded in 24-well tissue culture plate were fed with antibiotic-free low serum (0.5% FBS) Dulbecco's modified Eagle's medium for 18 h before transfection using Lipofectamine 2000 (Invitrogen). Low serum conditions were maintained throughout the experiment. In all, 293 cells were transfected with COX-2 full-length promoter construct (P2-1900-luc), along with increasing concentrations (0.5–3 μg) of either control pcDNA or v-FLIP/K13 expression plasmid. Forty-eight hours after transfection, cells were harvested, lysed and assayed for R.L.U. as described before. Promoter induction in control pcDNA cotransfected cells was considered as onefold. ( f ) A total of 293 cells were cotransfected with various COX-2 deletion constructs (as mentioned in the supplement) along with 2 μg each of control pcDNA or v-FLIP/K13 expression plasmid. Forty-eight hours after transfection, cells were harvested and assayed for R.L.U. as described earlier. ( g ) 293 cells were cotransfected with COX-2 promoter constructs (P2-1900 or P2-1102) along with 2 μg expression plasmids of pcDNA, IKK2DN, IκβM, p65 or <t>VIVIT.</t> Promoter induction in v-FLIP/K13-transfected cells was considered as 100%. Data in Figure 1E–G represent the mean R.L.U. after normalizing with the cotransfected Renilla activity. Each reaction was done in triplicate and each point represents the average±s.d. of three experiments. Promoter induction in pcDNA-transfected cells was considered as onefold. ( h ) pSIN or v-FLIP/K13-transduced HMVEC-d cells were untreated or treated with either DMSO solvent control or NF-κB inhibitor Bay 11-7082 for 1 h. Inhibitor or solvent control were removed after 1 h and cells were replenished with growth medium for 24 h or 48 h and COX-2 gene expression was measured. COX-2 gene expression in pSIN-HMVECs was considered as onefold. COX-2 gene expression in untreated v-FLIP/K13-HMVECs was considered as 100%. ( i ) v-FLIP transduction in HMVECs induces <t>NFAT</t> activation. Relative activation was calculated by taking the activation in pSIN-transduced cells as onefold at all of the indicated times. ( j ) v-FLIP transduction in HMVECs induces CREB activation (CREB serine 133 phosphorylation). Relative activation was calculated by taking the activation in p-SIN-HMVECs as onefold at the indicated times. * denotes statistically significant at P
    Vivit, supplied by Addgene inc, used in various techniques. Bioz Stars score: 88/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GenScript vivit
    <t>11R-VIVIT</t> inhibits pro-inflammatory cytokine expression in murine macrophages. BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. IL-12 p70 ( A ), IL-23 ( B ) and TNF ( C ) protein secretion were assayed from supernatants by ELISA. Each result represents mean ± SE of duplicate assays from three independent experiments. * p
    Vivit, supplied by GenScript, used in various techniques. Bioz Stars score: 88/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore cn vivit
    <t>11R-VIVIT</t> inhibits pro-inflammatory cytokine expression in murine macrophages. BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. IL-12 p70 ( A ), IL-23 ( B ) and TNF ( C ) protein secretion were assayed from supernatants by ELISA. Each result represents mean ± SE of duplicate assays from three independent experiments. * p
    Cn Vivit, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fisher Scientific vivit
    Calcineurin-NFAT-Dyrk1a signaling is implicated in harmine-Induced beta cell proliferation ( a ) A model depicting the calcium (Ca ++ )−calmodulin (CAM)−calcineurin (CnA and CnB subunits)−NFAT−c-MYC pathway to beta cell proliferation. The sites of action of harmine, <t>VIVIT</t> and <t>FK506</t> are shown in red. ( b ) Beta cell proliferation in dispersed human islets in response to harmine, in the presence and absence of VIVIT or FK506 ( n = four human preparations). ( c ) Adenoviral Dyrk1a overexpression in human islets, visualized by immunoblot. Representative of experiments from three islet preparations. ( d ) Ad. DYRK1A overexpression and immunolabeling for DYRK1A in human beta cells. Representative of experiments from three human islet preparations. ( e ) Effects of Ad. DYRK1A overexpression on harmine- and INDY-induced human beta cell proliferation. A minimum of 1000 beta cells was counted from four donors for each bar. ( f ) Effects of Ad.sh DYRK1A transduction in human islets. Representative of four human islet preparations. “Ad.Scr” indicates an adenovirus expressing scrambled shRNA. ( g ) An example of Ki67 immunolabeling in human beta cells transduced with Ad.sh DYRK1A . ( h ) Quantification of Ki67 in human beta cells transduced with Ad.sh DYRK1A . A minimum of 1000 beta cells was counted from five donors for each bar. ( i ) Effects of harmine (10 μM) or INDY (15 μM) treatment on NFAT4 translocation to the nucleus of human beta cells. Examples are shown in red arrows. Translocation of other NFATs is shown in Supplementary Figure 7 . In all relevant panels, the scale bar indicates 10 μm, error bars indicate s.e.m., and * indicates P
    Vivit, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 11r vivit
    Inhibition of Dengue Infection through Targeted Inhibition of ITAM Signaling Pathway Elements (A) A small molecule that targets Syk tyrosine kinase (Syk TK), PRT, inhibited dengue infection in FcγRNa + FcγRNIa + U937 monocytes. (B and C) A small-molecule inhibitor, INCA-6 (B), and a peptide inhibitor, <t>11R-VIVIT</t> (VIVIT) (C), which act by blocking calcineurin-substrate interactions at the calcineurin PxIxIT site, inhibited dengue infections. A scrambled peptide containing the amino acids found in VIVIT (11R VEET) did not inhibit infection (open circles). (D) Inhibition of dengue viruses (DENV2, DENV3), but not H1N1 or H3N2 influenza viruses (IAVs), was observed using the VIVIT inhibitor (solid lines); scrambled VEET peptide in dashed lines. (E) Treatment of cells with VIVIT up to 6 h after infection by dengue immune complexes significantly inhibited infection. (F) Model for susceptibility to clinically significant dengue infections in infants. Infants of mothers who are dengue-immune and have ≥10% afucosylated anti-E IgGs are at elevated risk for symptomatic primary dengue infections due to modulation of FcγRIIIa-expressing cells during infection. (G) Model for the roles of FcγRIIa, FcγRIIIa, and afucosylated dengue immune complexes in ADE. While FcγRIIa supports a majority of viral attachment and entry, FcγRIIIa signaling increases cellular ITAM signaling. ITAM signaling, in turn, triggers calcium flux and modulates the calcineurin signaling network, which supports dengue virus replication. *p
    11r Vivit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore vivit r
    Signal 1 determines resistance to ABT-737 in activated T cells. ( a ) Selective inhibitors of signal 1, 2 and 3 were added to an MLR during the stimulation phase with BM3.3 splenocytes reacting against CD8 T-cell-depleted B6 splenocytes to investigate the role of the different T-cell activation pathways for resistance to ABT-737(1 μ M, during additional 12 h of culture). The calcineurin inhibitor and signal 1 blocker CsA prevented resistance to ABT-737 in a concentration-dependent manner, whereas inhibition of signal 2 by CTLA4Ig and signal 3 by rapamycin did not influence this process. ( b ) The results obtained with CsA were confirmed by other inhibitors of this same pathway, namely the alternative calcineurin inhibitor tacrolimus and the NFAT inhibitor <t>VIVIT-R.</t> Cell viability of BM3.3 CD8 T cells was assessed by PI exclusion in FACS in at least three independent experiments. Percentage of cells treated with vehicle is given. Statistical comparison of ABT-737 versus vehicle: * P
    Vivit R, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Biomatik vivit peptide
    Signal 1 determines resistance to ABT-737 in activated T cells. ( a ) Selective inhibitors of signal 1, 2 and 3 were added to an MLR during the stimulation phase with BM3.3 splenocytes reacting against CD8 T-cell-depleted B6 splenocytes to investigate the role of the different T-cell activation pathways for resistance to ABT-737(1 μ M, during additional 12 h of culture). The calcineurin inhibitor and signal 1 blocker CsA prevented resistance to ABT-737 in a concentration-dependent manner, whereas inhibition of signal 2 by CTLA4Ig and signal 3 by rapamycin did not influence this process. ( b ) The results obtained with CsA were confirmed by other inhibitors of this same pathway, namely the alternative calcineurin inhibitor tacrolimus and the NFAT inhibitor <t>VIVIT-R.</t> Cell viability of BM3.3 CD8 T cells was assessed by PI exclusion in FACS in at least three independent experiments. Percentage of cells treated with vehicle is given. Statistical comparison of ABT-737 versus vehicle: * P
    Vivit Peptide, supplied by Biomatik, used in various techniques. Bioz Stars score: 88/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sigma-Genosys st vivit
    Anchored PKA and CaN have opposing effects on L-type Ca 2+ current in cultured hippocampal pyramidal neurons Peptides and other drugs were applied intracellularly via the patch pipet. Representative current recordings are shown for each time course as indicated; black records and colored records show currents 60 s (time point 1) and 250 s (time point 2) after establishing the whole cell recording configuration, respectively. All scale bars indicate 600 pA. ( A ) Average peak current time course during dialysis of intracellular recording solution alone (Con, ) or in the presence of either <t>VIVIT</t> ( ) or <t>Ht31</t> ( ). ( B ) The effect of CsA dialysis ( ) and co-application of VIVIT and PKI ( ). ( C and D ) as in A and B, but with FSK added to the intracellular recording solution.
    St Vivit, supplied by Sigma-Genosys, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sigma-Genosys 11r vivit peptide
    The mechanism of SLC4A2 in wear particle-induced osteoclast differentiation and function. (A) <t>11R-VIVIT,</t> an inhibitor of NFATc1 suppressed up-regulation of Slc4a2 expression during wear particle-induced osteoclastogenesis. (B) Expression of Nfatc1 was inhibited by knockdown of Slc4a2 with shRNA3 unexpectedly. (C–E) Suppression of osteoclast-specific gene expression ( Trap , Ctsk , Ctr ) with knockdown of Slc4a2 with shRNA3. (F) Knockdown of Slc4a2 , using shRNA3, inhibits F-actin ring formation in vitro . (G) The size of F-actin rings, measured using ImageJ ( ∗ P
    11r Vivit Peptide, supplied by Sigma-Genosys, used in various techniques. Bioz Stars score: 85/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore m 11r vivit
    The mechanism of SLC4A2 in wear particle-induced osteoclast differentiation and function. (A) <t>11R-VIVIT,</t> an inhibitor of NFATc1 suppressed up-regulation of Slc4a2 expression during wear particle-induced osteoclastogenesis. (B) Expression of Nfatc1 was inhibited by knockdown of Slc4a2 with shRNA3 unexpectedly. (C–E) Suppression of osteoclast-specific gene expression ( Trap , Ctsk , Ctr ) with knockdown of Slc4a2 with shRNA3. (F) Knockdown of Slc4a2 , using shRNA3, inhibits F-actin ring formation in vitro . (G) The size of F-actin rings, measured using ImageJ ( ∗ P
    M 11r Vivit, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc vivit plasmid
    The mechanism of SLC4A2 in wear particle-induced osteoclast differentiation and function. (A) <t>11R-VIVIT,</t> an inhibitor of NFATc1 suppressed up-regulation of Slc4a2 expression during wear particle-induced osteoclastogenesis. (B) Expression of Nfatc1 was inhibited by knockdown of Slc4a2 with shRNA3 unexpectedly. (C–E) Suppression of osteoclast-specific gene expression ( Trap , Ctsk , Ctr ) with knockdown of Slc4a2 with shRNA3. (F) Knockdown of Slc4a2 , using shRNA3, inhibits F-actin ring formation in vitro . (G) The size of F-actin rings, measured using ImageJ ( ∗ P
    Vivit Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Addgene inc nfat inhibitory peptide vivit gfp
    The mechanism of SLC4A2 in wear particle-induced osteoclast differentiation and function. (A) <t>11R-VIVIT,</t> an inhibitor of NFATc1 suppressed up-regulation of Slc4a2 expression during wear particle-induced osteoclastogenesis. (B) Expression of Nfatc1 was inhibited by knockdown of Slc4a2 with shRNA3 unexpectedly. (C–E) Suppression of osteoclast-specific gene expression ( Trap , Ctsk , Ctr ) with knockdown of Slc4a2 with shRNA3. (F) Knockdown of Slc4a2 , using shRNA3, inhibits F-actin ring formation in vitro . (G) The size of F-actin rings, measured using ImageJ ( ∗ P
    Nfat Inhibitory Peptide Vivit Gfp, supplied by Addgene inc, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore nfatc inhibitor 11r vivit
    Effects of intrathecal treatment with <t>11R-VIVIT</t> on the expression levels of NFATc4, CCR2, and BK α 1 in the DRG of nerve-injured rats. 11R-VIVIT (20 μ g, twice/day, n = 8) or saline ( n = 8) was injected for the first 5 days after nerve injury. The mRNA levels of NFATc4 (A), CCR2 (B), and BK α 1 (C) were quantified using real-time PCR from left L5 and L6 DRG tissues obtained 14 days after nerve injury ( n = 8 in each group). * P
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    Merck KGaA nfat inhibitor 11r vivit
    NFATs are relevant in human oligodendrocytes. a <t>NFAT</t> expression in human iOLs (grey bars) and NPCs (open bars) by qrtPCR after normalization to GAPDH . Average NFAT levels in NPCs were set to 1 ( NFATc1 : 1.4 ± 0.3; NFATc2 : 14.6 ± 1.9; NFATc3 : 1.6 ± 0.3 and NFATc4 : 0.9 ± 0.2). b Immunocytochemistry of MBP-positive (red) iOL with antibodies directed against NFATs (white in upper row, green in lower row); counterstaining with Hoechst dye (blue). c – g Analysis of differentiating iOL cultured 21 ( c , e , h ) or 35 days ( d , f , g , i , j ) in absence (Ctr, open bars) or presence of 0.1 (light grey bars) to 0.5 µM (dark grey bars) <t>VIVIT</t> ( c – g , n = 4) or 1 (light grey bars) to 10 µM (dark grey bars) FK506 ( h – j , n = 3–4). VIVIT/FK506 incubation was continuous ( c – f , h , i ) or from days 21–35 ( g , j ). Cultures were stained with anti-O4 (green, c ) and anti-MBP antibodies (green, d ). From staining and FACS analysis (see Suppl. Fig. 7c, d ) the fraction of O4-positive cells after 21 days ( e , h ) and MBP-positive cells after 35 days ( f , i ) was determined. For treatment during days 21–35, the fraction of FACS-sorted O4-positive cells was determined that had reached a MBP-positive stage ( g , j ). The relative number of marker-positive cells under control conditions was set to 1 and used to normalize in pairwise fashion (values: 1 for control, 0.97 ± 0.01 for 21 days 0.1 µM VIVIT, 0.68 ± 0.06 for 21 days 0.5 µM VIVIT, 0.86 ± 0.10 for 35 days 0.1 µM VIVIT, 0.60 ± 0.10 for 35 days 0.5 µM VIVIT, 0.82 ± 0.05 for 0.1 µM VIVIT from days 21–35, 0.86 ± 0.04 for 0.5 µM VIVIT from days 21–35, 0.99 ± 0.21 for 21 days 1 µM FK506, 0.39 ± 0.17 for 21 days 10 µM FK506, 1.12 ± 0.36 for 35 days 1 µM FK506, 0.55 ± 0.09 for 35 days 10 µM FK506, 0.83 ± 0.17 for 1 µM FK506 from days 21–35 and 0.86 ± 0.10 for 10 µM FK506 from days 21–35). k – n NFAT immunohistochemistry in human brain tissue. Antibodies directed against NFATc3 and NFATc4 (red in k and l , black in m ) were combined with anti-NOGOA antibodies (green in k and l , red in m ). Arrows mark double positive cells, arrowheads NOGOA-positive oligodendrocytes without NFATc4. n Percentage of NOGOA-positive oligodendrocytes with nuclear NFATc4 in periplaque white matter (86 ± 4%), active and demyelinating lesions (41 ± 8%), active and post-demyelinating lesions (41 ± 8%), active and remyelinating plaques (59 ± 19%), and control CNS tissue (99 ± 1%). o Model of oligodendroglial Nfat action. Statistical significance was determined by two-tailed Student’s t -test ( a ) or Bonferroni-corrected one-way ANOVA ( e – j , n ) (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). Scale bars, 50 µm
    Nfat Inhibitor 11r Vivit, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 92/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cayman Chemical nfat inhibitor 11r vivit
    NFATs are relevant in human oligodendrocytes. a <t>NFAT</t> expression in human iOLs (grey bars) and NPCs (open bars) by qrtPCR after normalization to GAPDH . Average NFAT levels in NPCs were set to 1 ( NFATc1 : 1.4 ± 0.3; NFATc2 : 14.6 ± 1.9; NFATc3 : 1.6 ± 0.3 and NFATc4 : 0.9 ± 0.2). b Immunocytochemistry of MBP-positive (red) iOL with antibodies directed against NFATs (white in upper row, green in lower row); counterstaining with Hoechst dye (blue). c – g Analysis of differentiating iOL cultured 21 ( c , e , h ) or 35 days ( d , f , g , i , j ) in absence (Ctr, open bars) or presence of 0.1 (light grey bars) to 0.5 µM (dark grey bars) <t>VIVIT</t> ( c – g , n = 4) or 1 (light grey bars) to 10 µM (dark grey bars) FK506 ( h – j , n = 3–4). VIVIT/FK506 incubation was continuous ( c – f , h , i ) or from days 21–35 ( g , j ). Cultures were stained with anti-O4 (green, c ) and anti-MBP antibodies (green, d ). From staining and FACS analysis (see Suppl. Fig. 7c, d ) the fraction of O4-positive cells after 21 days ( e , h ) and MBP-positive cells after 35 days ( f , i ) was determined. For treatment during days 21–35, the fraction of FACS-sorted O4-positive cells was determined that had reached a MBP-positive stage ( g , j ). The relative number of marker-positive cells under control conditions was set to 1 and used to normalize in pairwise fashion (values: 1 for control, 0.97 ± 0.01 for 21 days 0.1 µM VIVIT, 0.68 ± 0.06 for 21 days 0.5 µM VIVIT, 0.86 ± 0.10 for 35 days 0.1 µM VIVIT, 0.60 ± 0.10 for 35 days 0.5 µM VIVIT, 0.82 ± 0.05 for 0.1 µM VIVIT from days 21–35, 0.86 ± 0.04 for 0.5 µM VIVIT from days 21–35, 0.99 ± 0.21 for 21 days 1 µM FK506, 0.39 ± 0.17 for 21 days 10 µM FK506, 1.12 ± 0.36 for 35 days 1 µM FK506, 0.55 ± 0.09 for 35 days 10 µM FK506, 0.83 ± 0.17 for 1 µM FK506 from days 21–35 and 0.86 ± 0.10 for 10 µM FK506 from days 21–35). k – n NFAT immunohistochemistry in human brain tissue. Antibodies directed against NFATc3 and NFATc4 (red in k and l , black in m ) were combined with anti-NOGOA antibodies (green in k and l , red in m ). Arrows mark double positive cells, arrowheads NOGOA-positive oligodendrocytes without NFATc4. n Percentage of NOGOA-positive oligodendrocytes with nuclear NFATc4 in periplaque white matter (86 ± 4%), active and demyelinating lesions (41 ± 8%), active and post-demyelinating lesions (41 ± 8%), active and remyelinating plaques (59 ± 19%), and control CNS tissue (99 ± 1%). o Model of oligodendroglial Nfat action. Statistical significance was determined by two-tailed Student’s t -test ( a ) or Bonferroni-corrected one-way ANOVA ( e – j , n ) (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). Scale bars, 50 µm
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    Image Search Results


    Impairing calcineurin/NFAT signaling results in hair growth and stem cell proliferation (A) Schematic illustrating the experimental design of CSA experiments. (B-C) Quantification of anagen induction and BrdU incorporation in follicle stem cells of the bulge (Bu) with cyclosporine A (CSA) treatment. The anagen control used was the first hair cycle (P18-P25). Data are the mean ± SEM for 50−100 follicles for 3 individual mice for each timepoint. Asterisks in (C) indicate hair shaft autofluorescence. (D-E) Immunohistochemistry and western analysis of NFATc1 expression following 3d treatment with vehicle or CSA. (F) Colony formation and cell number of bulge cells following treatment with vehicle, CSA, 11R-VIVIT, or 11R-VEET. N= 3 experiments with independent sorted populations. (G) NFAT reporter activity in response to calcium ionophore (I), phorbol myristate acetate (PMA), caNFATc1, CSA or 11R-VIVIT. Data are mean ± SEM. N=3−5 individual experiments for each treatment. Asterisks indicate significance, p

    Journal: Cell

    Article Title: NFATc1 balances quiescence and proliferation of skin stem cells

    doi: 10.1016/j.cell.2007.11.047

    Figure Lengend Snippet: Impairing calcineurin/NFAT signaling results in hair growth and stem cell proliferation (A) Schematic illustrating the experimental design of CSA experiments. (B-C) Quantification of anagen induction and BrdU incorporation in follicle stem cells of the bulge (Bu) with cyclosporine A (CSA) treatment. The anagen control used was the first hair cycle (P18-P25). Data are the mean ± SEM for 50−100 follicles for 3 individual mice for each timepoint. Asterisks in (C) indicate hair shaft autofluorescence. (D-E) Immunohistochemistry and western analysis of NFATc1 expression following 3d treatment with vehicle or CSA. (F) Colony formation and cell number of bulge cells following treatment with vehicle, CSA, 11R-VIVIT, or 11R-VEET. N= 3 experiments with independent sorted populations. (G) NFAT reporter activity in response to calcium ionophore (I), phorbol myristate acetate (PMA), caNFATc1, CSA or 11R-VIVIT. Data are mean ± SEM. N=3−5 individual experiments for each treatment. Asterisks indicate significance, p

    Article Snippet: After the first passage, the MKs were treated with vehicle, cyclosporine A (1μM; Sigma), cell-permeable VIVIT (11R-VIVIT; 1μM; Calbiochem), or cell-permeable scrambled VIVIT, (11R-VEET; generous gift from Masayuki Matsushita and Rockefeller Proteomics Resource Center)( ) and analyzed for colony size ( > 5 cells) or cell number for the indicated days.

    Techniques: BrdU Incorporation Assay, Mouse Assay, Immunohistochemistry, Western Blot, Expressing, Activity Assay

    Sildenafil inhibits hypoxia-induced nuclear translocation of NFATc3 in cultured human PASMC . Human PASMC were cultured with SMBM (2% FBS) under nomoxia or hypoxia (3% O 2 ) in the presence of sildenafil (100 nM), 8-brom-cGMP (100 μM), SKF96365 (7.5 μM) or VIVIT (4 μM) respectively for 72 h. NFATc3 was determined by confocal microscopy of immunofluorescence. The primary antibody of NFATc3 was detected with Rhodamine (TRITC)-conjugated AffiniPure Goat Anti-mouse IgG (green). Slides were counterstained with nuclei dye hoechest33258 (blue). A: Immunofluorescence image of NFATc3 in human PASMC (×1000). B: The nuclear translocation of NFATc3 was calculated by comparing the ratio of nuclear NFATc3 immunofluorescence/total NFATc3 immunofluorescence. n = 20, ### P

    Journal: Respiratory Research

    Article Title: Inhibition of SOC/Ca2+/NFAT pathway is involved in the anti-proliferative effect of sildenafil on pulmonary artery smooth muscle cells

    doi: 10.1186/1465-9921-10-123

    Figure Lengend Snippet: Sildenafil inhibits hypoxia-induced nuclear translocation of NFATc3 in cultured human PASMC . Human PASMC were cultured with SMBM (2% FBS) under nomoxia or hypoxia (3% O 2 ) in the presence of sildenafil (100 nM), 8-brom-cGMP (100 μM), SKF96365 (7.5 μM) or VIVIT (4 μM) respectively for 72 h. NFATc3 was determined by confocal microscopy of immunofluorescence. The primary antibody of NFATc3 was detected with Rhodamine (TRITC)-conjugated AffiniPure Goat Anti-mouse IgG (green). Slides were counterstained with nuclei dye hoechest33258 (blue). A: Immunofluorescence image of NFATc3 in human PASMC (×1000). B: The nuclear translocation of NFATc3 was calculated by comparing the ratio of nuclear NFATc3 immunofluorescence/total NFATc3 immunofluorescence. n = 20, ### P

    Article Snippet: St. Louis, MO, USA), VIVIT (480401, Calbiochem, Darmstadt, Germany) and 8-brom-cGMP (Sigma-Aldrich.

    Techniques: Translocation Assay, Cell Culture, Confocal Microscopy, Immunofluorescence

    CyP facilitate HPV infection. 293TT (A), HaCaT (B), or HeLa cells (B) were infected with HPV16 (A,B,C) or HPV18 pseudovirus (B) in presence of indicated inhibitors and infection was scored at 72 hpi. (D) Effect of drugs on cell growth was determined by the MTT assay. We did not notice significant increase in cell death. CsA: cyclosporine A; VIVIT: cell permeable 11R-VIVIT; Nfdp: nifedipin; Vpml: verapamil. Representative graphs are based on three replicates each with standard deviation indicated.

    Journal: PLoS Pathogens

    Article Title: Target Cell Cyclophilins Facilitate Human Papillomavirus Type 16 Infection

    doi: 10.1371/journal.ppat.1000524

    Figure Lengend Snippet: CyP facilitate HPV infection. 293TT (A), HaCaT (B), or HeLa cells (B) were infected with HPV16 (A,B,C) or HPV18 pseudovirus (B) in presence of indicated inhibitors and infection was scored at 72 hpi. (D) Effect of drugs on cell growth was determined by the MTT assay. We did not notice significant increase in cell death. CsA: cyclosporine A; VIVIT: cell permeable 11R-VIVIT; Nfdp: nifedipin; Vpml: verapamil. Representative graphs are based on three replicates each with standard deviation indicated.

    Article Snippet: Verapamil (cat #: 676777), Nifedipine (cat #: 481981), 11R-VIVIT (cat #: 480401) and INCA-6 (cat #: 480403) were obtained from Calbiochem.

    Techniques: Infection, MTT Assay, Standard Deviation

    Regulation of IL2 by NFAT inferred by dynamic Boolean model: (a) network of interactions between IL2 and the TFs regulating its expression assembled from literature, (b) enhanced regulatory network inferred using dynamic Boolean model, and (c) the expression of IL2 mRNA evaluated by RT-PCR in DCs treated for one hour with medium containing the NFAT inhibitors CsA (CsA), VIVIT peptide (VIVIT), or vehicle (−), and then left mock-infected (mock) or infected with IV during 2 hours. The expression levels of IL2 in infected cells were normalized to the levels of nontreated, mock-infected cells.

    Journal: Computational and Mathematical Methods in Medicine

    Article Title: Boolean Modeling of Cellular and Molecular Pathways Involved in Influenza Infection

    doi: 10.1155/2016/7686081

    Figure Lengend Snippet: Regulation of IL2 by NFAT inferred by dynamic Boolean model: (a) network of interactions between IL2 and the TFs regulating its expression assembled from literature, (b) enhanced regulatory network inferred using dynamic Boolean model, and (c) the expression of IL2 mRNA evaluated by RT-PCR in DCs treated for one hour with medium containing the NFAT inhibitors CsA (CsA), VIVIT peptide (VIVIT), or vehicle (−), and then left mock-infected (mock) or infected with IV during 2 hours. The expression levels of IL2 in infected cells were normalized to the levels of nontreated, mock-infected cells.

    Article Snippet: To directly address the role of NFAT in IL2 signaling pathway, we treated IV-infected DCs that were treated with the two NFAT inhibitors, CsA, and the VIVIT peptide, which inhibit the phosphatase calcineurin, blocking NFAT activation [ , ].

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Infection

    Effects of 11R-VIVIT and NFAT2 gene knockdown on the expression of the uPA receptor (Plaur) gene in podocytes treated with high glucose. (A)The mRNA level of uPA receptors was examined using real-time quantitative RT-PCR analysis, and GAPDH mRNA was used as an internal control. Quantitative data were calculated by 2 -△△ CT . (B) The protein level of uPA receptors was analysed using Western blot analysis( n = 3). (C) Densitometry analysis of three repetitions of the experiment shown in (B). Note NG, normal glucose (5.3 mM) group; HG, high glucose group(30 mM); MA, normal glucose (5.3 mM) + mannitol(24.7 mM)group, as an osmolality control; HG+11R-VIVIT, High glucose (30 mM)+ 11R-VIVIT(100 nM, a cell permeable NFAT inhibitor) group; HG + NFAT2-siRNA, High glucose (30 mM) + NFAT2-siRNA (50 nM) group; HG + control-siRNA, High glucose (30 mM) + control-siRNA (50 nM), as a negative control group. All Values are expressed as the mean ± SEM. * P

    Journal: British Journal of Pharmacology

    Article Title: NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice

    doi: 10.1111/bph.12292

    Figure Lengend Snippet: Effects of 11R-VIVIT and NFAT2 gene knockdown on the expression of the uPA receptor (Plaur) gene in podocytes treated with high glucose. (A)The mRNA level of uPA receptors was examined using real-time quantitative RT-PCR analysis, and GAPDH mRNA was used as an internal control. Quantitative data were calculated by 2 -△△ CT . (B) The protein level of uPA receptors was analysed using Western blot analysis( n = 3). (C) Densitometry analysis of three repetitions of the experiment shown in (B). Note NG, normal glucose (5.3 mM) group; HG, high glucose group(30 mM); MA, normal glucose (5.3 mM) + mannitol(24.7 mM)group, as an osmolality control; HG+11R-VIVIT, High glucose (30 mM)+ 11R-VIVIT(100 nM, a cell permeable NFAT inhibitor) group; HG + NFAT2-siRNA, High glucose (30 mM) + NFAT2-siRNA (50 nM) group; HG + control-siRNA, High glucose (30 mM) + control-siRNA (50 nM), as a negative control group. All Values are expressed as the mean ± SEM. * P

    Article Snippet: At 12 weeks of age, half of the db/db mice were randomly chosen to receive 11R-VIVIT, a special inhibitor of NFAT (Merck KGaA, Darmstadt, Germany).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot, Negative Control

    Effects of 11R-VIVIT and NFAT2 gene knockdown on the expression of NFAT2 in podocytes treated with high glucose. (A) Confocal images of podocytes showing the expression of NFAT2 (red) and DAPI-stained nuclei (blue). (B) High glucose treatment increases the expression of NFAT2 detected by immunoblot in a nuclear extract of podocytes. Histone 3 was used as a nuclear protein control. (C) Densitometric analysis of three repetitions of the experiment shown in (C). Values are expressed as the mean ± SEM. * P

    Journal: British Journal of Pharmacology

    Article Title: NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice

    doi: 10.1111/bph.12292

    Figure Lengend Snippet: Effects of 11R-VIVIT and NFAT2 gene knockdown on the expression of NFAT2 in podocytes treated with high glucose. (A) Confocal images of podocytes showing the expression of NFAT2 (red) and DAPI-stained nuclei (blue). (B) High glucose treatment increases the expression of NFAT2 detected by immunoblot in a nuclear extract of podocytes. Histone 3 was used as a nuclear protein control. (C) Densitometric analysis of three repetitions of the experiment shown in (C). Values are expressed as the mean ± SEM. * P

    Article Snippet: At 12 weeks of age, half of the db/db mice were randomly chosen to receive 11R-VIVIT, a special inhibitor of NFAT (Merck KGaA, Darmstadt, Germany).

    Techniques: Expressing, Staining

    11R-VIVIT attenuates the high glucose-induced dysfunction of the filtration barrier in podocytes. Graphic presentation shows the albumin flux rate across the differentiated podocyte monolayer in each group after 6 h of treatment. Values (mg·mL −1 ) are means ± SEM. * P

    Journal: British Journal of Pharmacology

    Article Title: NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice

    doi: 10.1111/bph.12292

    Figure Lengend Snippet: 11R-VIVIT attenuates the high glucose-induced dysfunction of the filtration barrier in podocytes. Graphic presentation shows the albumin flux rate across the differentiated podocyte monolayer in each group after 6 h of treatment. Values (mg·mL −1 ) are means ± SEM. * P

    Article Snippet: At 12 weeks of age, half of the db/db mice were randomly chosen to receive 11R-VIVIT, a special inhibitor of NFAT (Merck KGaA, Darmstadt, Germany).

    Techniques: Filtration

    The proposed mechanism for 11R-VIVIT-induced protection against podocyte injury of diabetic nephropathy in db/db mice. Solid arrows represent steps supported by the data from the present study, whereas dashed arrows represent steps supported by our previously published data.

    Journal: British Journal of Pharmacology

    Article Title: NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice

    doi: 10.1111/bph.12292

    Figure Lengend Snippet: The proposed mechanism for 11R-VIVIT-induced protection against podocyte injury of diabetic nephropathy in db/db mice. Solid arrows represent steps supported by the data from the present study, whereas dashed arrows represent steps supported by our previously published data.

    Article Snippet: At 12 weeks of age, half of the db/db mice were randomly chosen to receive 11R-VIVIT, a special inhibitor of NFAT (Merck KGaA, Darmstadt, Germany).

    Techniques: Mouse Assay

    NFAT inhibitor (11R-VIVIT) inhibited NFAT2 activation and uPA receptor expression in glomerular podocytes. (A) Double immunofluorescent staining of NFAT2 (green), WT1 identified podocytes (red), and merged images in the kidney from non-diabetic mice or diabetic animals. Although NFAT2 was faintly observed in non-diabetic glomeruli, increased immunoreactivity for NFAT2 was observed in the diabetic mice. Co-staining for NFAT2 (green) demonstrates that NFAT2 activation is markedly inhibited in glomerular podocytes of 11R-VIVIT-treated diabetic mice. Original magnification ×400. (B) Double immunofluorescent staining of uPA receptors (red), synaptopodin identified podocytes (green), and merged images (yellow) in the kidney from non-diabetic mice or diabetic animals. The protein expression of uPA receptors (red) was significantly increased in diabetic glomeruli. Co-staining for uPA receptors (red) demonstrates that uPA receptor expression was restored in glomerular podocytes of 11R-VIVIT treated diabetic mice, original magnification ×400. (C) Double immunofluorescent staining of uPA receptors (red), synaptopodin identified podocytes (green), and merged images in the kidney from patients with DN or healthy control (normal renal tissues from patients with renal cell carcinoma). Co-staining for uPA receptors (red) demonstrates that their expression was markedly increased in glomerular podocytes of DN patients. Original magnification ×200.

    Journal: British Journal of Pharmacology

    Article Title: NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice

    doi: 10.1111/bph.12292

    Figure Lengend Snippet: NFAT inhibitor (11R-VIVIT) inhibited NFAT2 activation and uPA receptor expression in glomerular podocytes. (A) Double immunofluorescent staining of NFAT2 (green), WT1 identified podocytes (red), and merged images in the kidney from non-diabetic mice or diabetic animals. Although NFAT2 was faintly observed in non-diabetic glomeruli, increased immunoreactivity for NFAT2 was observed in the diabetic mice. Co-staining for NFAT2 (green) demonstrates that NFAT2 activation is markedly inhibited in glomerular podocytes of 11R-VIVIT-treated diabetic mice. Original magnification ×400. (B) Double immunofluorescent staining of uPA receptors (red), synaptopodin identified podocytes (green), and merged images (yellow) in the kidney from non-diabetic mice or diabetic animals. The protein expression of uPA receptors (red) was significantly increased in diabetic glomeruli. Co-staining for uPA receptors (red) demonstrates that uPA receptor expression was restored in glomerular podocytes of 11R-VIVIT treated diabetic mice, original magnification ×400. (C) Double immunofluorescent staining of uPA receptors (red), synaptopodin identified podocytes (green), and merged images in the kidney from patients with DN or healthy control (normal renal tissues from patients with renal cell carcinoma). Co-staining for uPA receptors (red) demonstrates that their expression was markedly increased in glomerular podocytes of DN patients. Original magnification ×200.

    Article Snippet: At 12 weeks of age, half of the db/db mice were randomly chosen to receive 11R-VIVIT, a special inhibitor of NFAT (Merck KGaA, Darmstadt, Germany).

    Techniques: Activation Assay, Expressing, Staining, Mouse Assay

    Effect of urinary albumin excretion and kidney function in db/db mice. The graphs show urinary albumin excretion (μg·mg −1 creatinine) (A), creatinine clearance (B) in vehicle-treated BKS mice, vehicle treated db/db mice and 11R-VIVIT treated db/db mice. A 24-h pooled urine sample was collected in a metabolic cage at 12, 14, 16, 18 and 20 weeks of age for each mouse. Urinary albumin or urinary and serum creatinine were measured using a competitive ELISA. Data are shown as means ± SEM. n = 5. * P

    Journal: British Journal of Pharmacology

    Article Title: NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice

    doi: 10.1111/bph.12292

    Figure Lengend Snippet: Effect of urinary albumin excretion and kidney function in db/db mice. The graphs show urinary albumin excretion (μg·mg −1 creatinine) (A), creatinine clearance (B) in vehicle-treated BKS mice, vehicle treated db/db mice and 11R-VIVIT treated db/db mice. A 24-h pooled urine sample was collected in a metabolic cage at 12, 14, 16, 18 and 20 weeks of age for each mouse. Urinary albumin or urinary and serum creatinine were measured using a competitive ELISA. Data are shown as means ± SEM. n = 5. * P

    Article Snippet: At 12 weeks of age, half of the db/db mice were randomly chosen to receive 11R-VIVIT, a special inhibitor of NFAT (Merck KGaA, Darmstadt, Germany).

    Techniques: Mouse Assay, Competitive ELISA

    11R-VIVIT partially restores podocyte number. (A) Typical images of WT1 stained glomeruli from BKS , db/db and db/db +11R-VIVIT mice. Arrows point to WT-1-positive cells. Magnification ×400. (B) Quantification of podocyte markers WT-1-positive cells per glomerular section. The number of WT1-stained nuclei was obtained from 20 glomeruli per kidney section from five mice per group. Data are means ± SEM. * P

    Journal: British Journal of Pharmacology

    Article Title: NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice

    doi: 10.1111/bph.12292

    Figure Lengend Snippet: 11R-VIVIT partially restores podocyte number. (A) Typical images of WT1 stained glomeruli from BKS , db/db and db/db +11R-VIVIT mice. Arrows point to WT-1-positive cells. Magnification ×400. (B) Quantification of podocyte markers WT-1-positive cells per glomerular section. The number of WT1-stained nuclei was obtained from 20 glomeruli per kidney section from five mice per group. Data are means ± SEM. * P

    Article Snippet: At 12 weeks of age, half of the db/db mice were randomly chosen to receive 11R-VIVIT, a special inhibitor of NFAT (Merck KGaA, Darmstadt, Germany).

    Techniques: Staining, Mouse Assay

    11R-VIVIT attenuated glomerular basement membrane (GBM) thickening and podocyte foot process effacement. Kidney samples from non-diabetic BKS control mice treated with PBS and db/db mice treated with PBS or 11R-VIVIT were subjected to electron microscopic analyses. (A) Representative electron micrographs show GBM thickening and foot process effacement in the PBS treated diabetic kidney. The morphological injury was attenuated in diabetic mice that received 11R-VIVIT. Scale bar, 1000 nm. (B) Effect of the different treatments on the thickness of the GBM. Data are mean ± SEM. # P

    Journal: British Journal of Pharmacology

    Article Title: NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice

    doi: 10.1111/bph.12292

    Figure Lengend Snippet: 11R-VIVIT attenuated glomerular basement membrane (GBM) thickening and podocyte foot process effacement. Kidney samples from non-diabetic BKS control mice treated with PBS and db/db mice treated with PBS or 11R-VIVIT were subjected to electron microscopic analyses. (A) Representative electron micrographs show GBM thickening and foot process effacement in the PBS treated diabetic kidney. The morphological injury was attenuated in diabetic mice that received 11R-VIVIT. Scale bar, 1000 nm. (B) Effect of the different treatments on the thickness of the GBM. Data are mean ± SEM. # P

    Article Snippet: At 12 weeks of age, half of the db/db mice were randomly chosen to receive 11R-VIVIT, a special inhibitor of NFAT (Merck KGaA, Darmstadt, Germany).

    Techniques: Mouse Assay

    Effects of 11R-VIVIT on mesangial expansion. (A) Representative photomicrographs of periodic acid–Schiff staining of glomeruli of BKS + PBS, db/db + PBS, db/db +11R-VIVIT mice at 20 weeks. (B) Semiquantitative analyses of mesangial expansion at 20 weeks ( n = 5). Original magnification ×400. Data are mean ± SEM. * P

    Journal: British Journal of Pharmacology

    Article Title: NFAT2 inhibitor ameliorates diabetic nephropathy and podocyte injury in db/db mice

    doi: 10.1111/bph.12292

    Figure Lengend Snippet: Effects of 11R-VIVIT on mesangial expansion. (A) Representative photomicrographs of periodic acid–Schiff staining of glomeruli of BKS + PBS, db/db + PBS, db/db +11R-VIVIT mice at 20 weeks. (B) Semiquantitative analyses of mesangial expansion at 20 weeks ( n = 5). Original magnification ×400. Data are mean ± SEM. * P

    Article Snippet: At 12 weeks of age, half of the db/db mice were randomly chosen to receive 11R-VIVIT, a special inhibitor of NFAT (Merck KGaA, Darmstadt, Germany).

    Techniques: Staining, Mouse Assay

    Effect of v-FLIP/K13 on COX-2, mPGES-1 gene expression, COX-2 and COX-1 protein levels and PGE2 secretion and role of cis -acting factors on transcriptional regulation of COX-2 promoter by v-FLIP/K13. ( a , b ) HMVEC-d cells were infected with KSHV latency gene (LANA-1, v-FLIP, v-Cyclin, K8 and K12), GFP or empty vector (pSIN) expressing lentiviruses. Forty-eight hours after infection, cells were washed, lysed, total RNA was prepared, and COX-2 and mPGES-1 expression were quantitated using COX-2 and mPGES-1 gene-specific primers normalized to GAPDH levels as described earlier. 5 Fold induction was calculated by considering levels in pSIN lentivirus-infected cells as onefold. ( c ) Supernatants collected from cells in ( a ) were used for PGE2 detection by methods described previously. 5 Each reaction was done in triplicate and each point represents the average±s.d. of three independent experiments. ( d ) Lysates prepared from the above mentioned cells were used for quantitating COX-2 protein levels. ( e ) A total of 293 cells seeded in 24-well tissue culture plate were fed with antibiotic-free low serum (0.5% FBS) Dulbecco's modified Eagle's medium for 18 h before transfection using Lipofectamine 2000 (Invitrogen). Low serum conditions were maintained throughout the experiment. In all, 293 cells were transfected with COX-2 full-length promoter construct (P2-1900-luc), along with increasing concentrations (0.5–3 μg) of either control pcDNA or v-FLIP/K13 expression plasmid. Forty-eight hours after transfection, cells were harvested, lysed and assayed for R.L.U. as described before. Promoter induction in control pcDNA cotransfected cells was considered as onefold. ( f ) A total of 293 cells were cotransfected with various COX-2 deletion constructs (as mentioned in the supplement) along with 2 μg each of control pcDNA or v-FLIP/K13 expression plasmid. Forty-eight hours after transfection, cells were harvested and assayed for R.L.U. as described earlier. ( g ) 293 cells were cotransfected with COX-2 promoter constructs (P2-1900 or P2-1102) along with 2 μg expression plasmids of pcDNA, IKK2DN, IκβM, p65 or VIVIT. Promoter induction in v-FLIP/K13-transfected cells was considered as 100%. Data in Figure 1E–G represent the mean R.L.U. after normalizing with the cotransfected Renilla activity. Each reaction was done in triplicate and each point represents the average±s.d. of three experiments. Promoter induction in pcDNA-transfected cells was considered as onefold. ( h ) pSIN or v-FLIP/K13-transduced HMVEC-d cells were untreated or treated with either DMSO solvent control or NF-κB inhibitor Bay 11-7082 for 1 h. Inhibitor or solvent control were removed after 1 h and cells were replenished with growth medium for 24 h or 48 h and COX-2 gene expression was measured. COX-2 gene expression in pSIN-HMVECs was considered as onefold. COX-2 gene expression in untreated v-FLIP/K13-HMVECs was considered as 100%. ( i ) v-FLIP transduction in HMVECs induces NFAT activation. Relative activation was calculated by taking the activation in pSIN-transduced cells as onefold at all of the indicated times. ( j ) v-FLIP transduction in HMVECs induces CREB activation (CREB serine 133 phosphorylation). Relative activation was calculated by taking the activation in p-SIN-HMVECs as onefold at the indicated times. * denotes statistically significant at P

    Journal: Oncogenesis

    Article Title: COX-2/PGE2: molecular ambassadors of Kaposi's sarcoma-associated herpes virus oncoprotein-v-FLIP

    doi: 10.1038/oncsis.2012.5

    Figure Lengend Snippet: Effect of v-FLIP/K13 on COX-2, mPGES-1 gene expression, COX-2 and COX-1 protein levels and PGE2 secretion and role of cis -acting factors on transcriptional regulation of COX-2 promoter by v-FLIP/K13. ( a , b ) HMVEC-d cells were infected with KSHV latency gene (LANA-1, v-FLIP, v-Cyclin, K8 and K12), GFP or empty vector (pSIN) expressing lentiviruses. Forty-eight hours after infection, cells were washed, lysed, total RNA was prepared, and COX-2 and mPGES-1 expression were quantitated using COX-2 and mPGES-1 gene-specific primers normalized to GAPDH levels as described earlier. 5 Fold induction was calculated by considering levels in pSIN lentivirus-infected cells as onefold. ( c ) Supernatants collected from cells in ( a ) were used for PGE2 detection by methods described previously. 5 Each reaction was done in triplicate and each point represents the average±s.d. of three independent experiments. ( d ) Lysates prepared from the above mentioned cells were used for quantitating COX-2 protein levels. ( e ) A total of 293 cells seeded in 24-well tissue culture plate were fed with antibiotic-free low serum (0.5% FBS) Dulbecco's modified Eagle's medium for 18 h before transfection using Lipofectamine 2000 (Invitrogen). Low serum conditions were maintained throughout the experiment. In all, 293 cells were transfected with COX-2 full-length promoter construct (P2-1900-luc), along with increasing concentrations (0.5–3 μg) of either control pcDNA or v-FLIP/K13 expression plasmid. Forty-eight hours after transfection, cells were harvested, lysed and assayed for R.L.U. as described before. Promoter induction in control pcDNA cotransfected cells was considered as onefold. ( f ) A total of 293 cells were cotransfected with various COX-2 deletion constructs (as mentioned in the supplement) along with 2 μg each of control pcDNA or v-FLIP/K13 expression plasmid. Forty-eight hours after transfection, cells were harvested and assayed for R.L.U. as described earlier. ( g ) 293 cells were cotransfected with COX-2 promoter constructs (P2-1900 or P2-1102) along with 2 μg expression plasmids of pcDNA, IKK2DN, IκβM, p65 or VIVIT. Promoter induction in v-FLIP/K13-transfected cells was considered as 100%. Data in Figure 1E–G represent the mean R.L.U. after normalizing with the cotransfected Renilla activity. Each reaction was done in triplicate and each point represents the average±s.d. of three experiments. Promoter induction in pcDNA-transfected cells was considered as onefold. ( h ) pSIN or v-FLIP/K13-transduced HMVEC-d cells were untreated or treated with either DMSO solvent control or NF-κB inhibitor Bay 11-7082 for 1 h. Inhibitor or solvent control were removed after 1 h and cells were replenished with growth medium for 24 h or 48 h and COX-2 gene expression was measured. COX-2 gene expression in pSIN-HMVECs was considered as onefold. COX-2 gene expression in untreated v-FLIP/K13-HMVECs was considered as 100%. ( i ) v-FLIP transduction in HMVECs induces NFAT activation. Relative activation was calculated by taking the activation in pSIN-transduced cells as onefold at all of the indicated times. ( j ) v-FLIP transduction in HMVECs induces CREB activation (CREB serine 133 phosphorylation). Relative activation was calculated by taking the activation in p-SIN-HMVECs as onefold at the indicated times. * denotes statistically significant at P

    Article Snippet: VIVIT, a plasmid containing the NFAT regulatory domain, was obtained from Addgene (Cambridge, MA, USA) and has been described previously.

    Techniques: Expressing, Infection, Plasmid Preparation, Modification, Transfection, Construct, Activity Assay, Transduction, Activation Assay

    11R-VIVIT inhibits pro-inflammatory cytokine expression in murine macrophages. BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. IL-12 p70 ( A ), IL-23 ( B ) and TNF ( C ) protein secretion were assayed from supernatants by ELISA. Each result represents mean ± SE of duplicate assays from three independent experiments. * p

    Journal: PLoS ONE

    Article Title: A Cell Permeable Peptide Inhibitor of NFAT Inhibits Macrophage Cytokine Expression and Ameliorates Experimental Colitis

    doi: 10.1371/journal.pone.0034172

    Figure Lengend Snippet: 11R-VIVIT inhibits pro-inflammatory cytokine expression in murine macrophages. BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. IL-12 p70 ( A ), IL-23 ( B ) and TNF ( C ) protein secretion were assayed from supernatants by ELISA. Each result represents mean ± SE of duplicate assays from three independent experiments. * p

    Article Snippet: 11R-VIVIT from EMD Bioscience will be referred to as VIVITEMD since this VIVIT was active at significantly lower concentrations compared to the VIVIT from GenScript.

    Techniques: Expressing, Mouse Assay, Enzyme-linked Immunosorbent Assay

    NFAT binds to Nos2 promoter and its selective inhibition abrogates nitric oxide secretion in macrophages. ( A ) Nuclear extracts were prepared from BMDMs stimulated with LPS (lane 1) or LPS and IFN-γ (lanes 2–7). Extracts were either incubated with a labeled probe NFAT/ISRE (element from region II of the promoter scheme), a competitor ISRE oligonucleotide, or with the indicated antibodies. DNA-protein complexes (indicated by arrow) were separated by electrophoresis. EMSA revealed binding of both IRF8 and NFATc1 to the same Nos2 promoter element. ( B ) BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. Nitric oxide secretion was assayed from supernatants by Greiss reaction. Experiments were performed in duplicate and repeated three times (mean ± SEM). ( C ) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with LPS alone or IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for Nos2 mRNA levels by real-time RT PCR. Data is representative of three independent experiments. * p

    Journal: PLoS ONE

    Article Title: A Cell Permeable Peptide Inhibitor of NFAT Inhibits Macrophage Cytokine Expression and Ameliorates Experimental Colitis

    doi: 10.1371/journal.pone.0034172

    Figure Lengend Snippet: NFAT binds to Nos2 promoter and its selective inhibition abrogates nitric oxide secretion in macrophages. ( A ) Nuclear extracts were prepared from BMDMs stimulated with LPS (lane 1) or LPS and IFN-γ (lanes 2–7). Extracts were either incubated with a labeled probe NFAT/ISRE (element from region II of the promoter scheme), a competitor ISRE oligonucleotide, or with the indicated antibodies. DNA-protein complexes (indicated by arrow) were separated by electrophoresis. EMSA revealed binding of both IRF8 and NFATc1 to the same Nos2 promoter element. ( B ) BMDMs from WT mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide for 1 h followed by LPS (100 ng/ml) and IFN-γ (10 ng/ml) for 24 h. Nitric oxide secretion was assayed from supernatants by Greiss reaction. Experiments were performed in duplicate and repeated three times (mean ± SEM). ( C ) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with LPS alone or IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for Nos2 mRNA levels by real-time RT PCR. Data is representative of three independent experiments. * p

    Article Snippet: 11R-VIVIT from EMD Bioscience will be referred to as VIVITEMD since this VIVIT was active at significantly lower concentrations compared to the VIVIT from GenScript.

    Techniques: Inhibition, Incubation, Labeling, Electrophoresis, Binding Assay, Mouse Assay, Quantitative RT-PCR

    11R-VIVIT inhibits IL-12 p40 protein and mRNA expression. ( A ) Murine bone marrow-derived macrophages (BMDMs) were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide 11R-VEET for 1 h followed by LPS (100 ng/ml) alone or together with IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. ( B ) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for Il12b mRNA levels by real-time RT-PCR. Results were normalized with the respect to the levels of β-actin mRNA, and represent the mean ± SE for duplicate assays from three independent experiments. * p

    Journal: PLoS ONE

    Article Title: A Cell Permeable Peptide Inhibitor of NFAT Inhibits Macrophage Cytokine Expression and Ameliorates Experimental Colitis

    doi: 10.1371/journal.pone.0034172

    Figure Lengend Snippet: 11R-VIVIT inhibits IL-12 p40 protein and mRNA expression. ( A ) Murine bone marrow-derived macrophages (BMDMs) were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or the control peptide 11R-VEET for 1 h followed by LPS (100 ng/ml) alone or together with IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. ( B ) BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT for 1 h followed by 1 h treatment with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) treatment for 4 h. Cells were harvested and total RNA was assayed for Il12b mRNA levels by real-time RT-PCR. Results were normalized with the respect to the levels of β-actin mRNA, and represent the mean ± SE for duplicate assays from three independent experiments. * p

    Article Snippet: 11R-VIVIT from EMD Bioscience will be referred to as VIVITEMD since this VIVIT was active at significantly lower concentrations compared to the VIVIT from GenScript.

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

    11R-VIVIT reduces DNA binding to the NFAT/IRF8 site in the murine IL-12 p40 promoter. BMDMs were either untreated (lane 1) or pretreated with the indicated concentrations of 11R-VEET (lanes 2–4) or 11R-VIVIT (lanes 5–7) for 1 h. Cells were then treated for 1 h with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) stimulation for 4 h. Cells were harvested and nuclear extracts were prepared for EMSA. 32 P labeled oligonucleotide probe spanning the NFAT/IRF8 site of the IL-12 p40 promoter [10] was incubated with 10 mg of nuclear extracts on ice for 30 min prior to electrophoresis. For supershift assays (lanes 3,4 and 6,7), 10 mg nuclear extract were incubated with 3 mg of anti-NFAT c1 (lanes 3 and 6) or anti-IRF8 (lanes 4 and 7) antibodies for 30 min on ice prior to addition of the 32 P labeled probe and 30 min incubation on ice followed by electrophoresis. The arrows represent DNA-protein complexes formed before and after incubation with the indicated antibodies. The above result is representative of five independent experiments.

    Journal: PLoS ONE

    Article Title: A Cell Permeable Peptide Inhibitor of NFAT Inhibits Macrophage Cytokine Expression and Ameliorates Experimental Colitis

    doi: 10.1371/journal.pone.0034172

    Figure Lengend Snippet: 11R-VIVIT reduces DNA binding to the NFAT/IRF8 site in the murine IL-12 p40 promoter. BMDMs were either untreated (lane 1) or pretreated with the indicated concentrations of 11R-VEET (lanes 2–4) or 11R-VIVIT (lanes 5–7) for 1 h. Cells were then treated for 1 h with IFN-γ (10 ng/ml) prior to LPS (100 ng/ml) stimulation for 4 h. Cells were harvested and nuclear extracts were prepared for EMSA. 32 P labeled oligonucleotide probe spanning the NFAT/IRF8 site of the IL-12 p40 promoter [10] was incubated with 10 mg of nuclear extracts on ice for 30 min prior to electrophoresis. For supershift assays (lanes 3,4 and 6,7), 10 mg nuclear extract were incubated with 3 mg of anti-NFAT c1 (lanes 3 and 6) or anti-IRF8 (lanes 4 and 7) antibodies for 30 min on ice prior to addition of the 32 P labeled probe and 30 min incubation on ice followed by electrophoresis. The arrows represent DNA-protein complexes formed before and after incubation with the indicated antibodies. The above result is representative of five independent experiments.

    Article Snippet: 11R-VIVIT from EMD Bioscience will be referred to as VIVITEMD since this VIVIT was active at significantly lower concentrations compared to the VIVIT from GenScript.

    Techniques: Binding Assay, Labeling, Incubation, Electrophoresis

    Inhibition of IL-12 p40 by 11R-VIVIT is independent of IL-10. ( A ) Murine BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT (white bars) or 11R-VEET (black bars) for 1 h followed by LPS (100 ng/ml) alone or together with IFN-γ (10 ng/ml) for 24 h. IL-10 protein secretion was assayed from supernatants by ELISA. ( B ) BMDMs from Il10 −/− mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and LPS plus IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. Results represent the mean ± SD for duplicate assays from three independent experiments. * p

    Journal: PLoS ONE

    Article Title: A Cell Permeable Peptide Inhibitor of NFAT Inhibits Macrophage Cytokine Expression and Ameliorates Experimental Colitis

    doi: 10.1371/journal.pone.0034172

    Figure Lengend Snippet: Inhibition of IL-12 p40 by 11R-VIVIT is independent of IL-10. ( A ) Murine BMDMs were either untreated or pretreated with the indicated concentrations of 11R-VIVIT (white bars) or 11R-VEET (black bars) for 1 h followed by LPS (100 ng/ml) alone or together with IFN-γ (10 ng/ml) for 24 h. IL-10 protein secretion was assayed from supernatants by ELISA. ( B ) BMDMs from Il10 −/− mice were either untreated or pretreated with the indicated concentrations of 11R-VIVIT or 11R-VEET for 1 h followed by LPS (100 ng/ml) and LPS plus IFN-γ (10 ng/ml) for 24 h. IL-12 p40 protein secretion was assayed from supernatants by ELISA. Results represent the mean ± SD for duplicate assays from three independent experiments. * p

    Article Snippet: 11R-VIVIT from EMD Bioscience will be referred to as VIVITEMD since this VIVIT was active at significantly lower concentrations compared to the VIVIT from GenScript.

    Techniques: Inhibition, Enzyme-linked Immunosorbent Assay, Mouse Assay

    Calcineurin-NFAT-Dyrk1a signaling is implicated in harmine-Induced beta cell proliferation ( a ) A model depicting the calcium (Ca ++ )−calmodulin (CAM)−calcineurin (CnA and CnB subunits)−NFAT−c-MYC pathway to beta cell proliferation. The sites of action of harmine, VIVIT and FK506 are shown in red. ( b ) Beta cell proliferation in dispersed human islets in response to harmine, in the presence and absence of VIVIT or FK506 ( n = four human preparations). ( c ) Adenoviral Dyrk1a overexpression in human islets, visualized by immunoblot. Representative of experiments from three islet preparations. ( d ) Ad. DYRK1A overexpression and immunolabeling for DYRK1A in human beta cells. Representative of experiments from three human islet preparations. ( e ) Effects of Ad. DYRK1A overexpression on harmine- and INDY-induced human beta cell proliferation. A minimum of 1000 beta cells was counted from four donors for each bar. ( f ) Effects of Ad.sh DYRK1A transduction in human islets. Representative of four human islet preparations. “Ad.Scr” indicates an adenovirus expressing scrambled shRNA. ( g ) An example of Ki67 immunolabeling in human beta cells transduced with Ad.sh DYRK1A . ( h ) Quantification of Ki67 in human beta cells transduced with Ad.sh DYRK1A . A minimum of 1000 beta cells was counted from five donors for each bar. ( i ) Effects of harmine (10 μM) or INDY (15 μM) treatment on NFAT4 translocation to the nucleus of human beta cells. Examples are shown in red arrows. Translocation of other NFATs is shown in Supplementary Figure 7 . In all relevant panels, the scale bar indicates 10 μm, error bars indicate s.e.m., and * indicates P

    Journal: Nature medicine

    Article Title: Induction of human pancreatic beta cell replication by inhibitors of dual specificity tyrosine regulated kinase

    doi: 10.1038/nm.3820

    Figure Lengend Snippet: Calcineurin-NFAT-Dyrk1a signaling is implicated in harmine-Induced beta cell proliferation ( a ) A model depicting the calcium (Ca ++ )−calmodulin (CAM)−calcineurin (CnA and CnB subunits)−NFAT−c-MYC pathway to beta cell proliferation. The sites of action of harmine, VIVIT and FK506 are shown in red. ( b ) Beta cell proliferation in dispersed human islets in response to harmine, in the presence and absence of VIVIT or FK506 ( n = four human preparations). ( c ) Adenoviral Dyrk1a overexpression in human islets, visualized by immunoblot. Representative of experiments from three islet preparations. ( d ) Ad. DYRK1A overexpression and immunolabeling for DYRK1A in human beta cells. Representative of experiments from three human islet preparations. ( e ) Effects of Ad. DYRK1A overexpression on harmine- and INDY-induced human beta cell proliferation. A minimum of 1000 beta cells was counted from four donors for each bar. ( f ) Effects of Ad.sh DYRK1A transduction in human islets. Representative of four human islet preparations. “Ad.Scr” indicates an adenovirus expressing scrambled shRNA. ( g ) An example of Ki67 immunolabeling in human beta cells transduced with Ad.sh DYRK1A . ( h ) Quantification of Ki67 in human beta cells transduced with Ad.sh DYRK1A . A minimum of 1000 beta cells was counted from five donors for each bar. ( i ) Effects of harmine (10 μM) or INDY (15 μM) treatment on NFAT4 translocation to the nucleus of human beta cells. Examples are shown in red arrows. Translocation of other NFATs is shown in Supplementary Figure 7 . In all relevant panels, the scale bar indicates 10 μm, error bars indicate s.e.m., and * indicates P

    Article Snippet: Reagents Reagents were as follows: INDY (4997, Tocris Biosciences), BrdU (RPN20, GE Healthcare), harmaline (51330, Sigma), harmane (103276, Sigma), harmalol (H125, Sigma), harmine (286044, Sigma, for in vitro studies), harmine hydrochloride (CAS 343-27-1, Santa Cruz, for in vivo studies), VIVIT (502306392, Fisher scientific), FK506 (tlrl-fk5, Invivogen), 1RH (10058, 475956, Calbiochem), etoposide (E1383, Sigma), recombinant human IL1-β (201-lb-005, R & D Systems), recombinant human TNF-α (210-TA-010, R & D Systems), WS6 (M60097-2s, Xcess Biosciences).

    Techniques: Chick Chorioallantoic Membrane Assay, Over Expression, Immunolabeling, Transduction, Expressing, shRNA, Translocation Assay

    Inhibition of Dengue Infection through Targeted Inhibition of ITAM Signaling Pathway Elements (A) A small molecule that targets Syk tyrosine kinase (Syk TK), PRT, inhibited dengue infection in FcγRNa + FcγRNIa + U937 monocytes. (B and C) A small-molecule inhibitor, INCA-6 (B), and a peptide inhibitor, 11R-VIVIT (VIVIT) (C), which act by blocking calcineurin-substrate interactions at the calcineurin PxIxIT site, inhibited dengue infections. A scrambled peptide containing the amino acids found in VIVIT (11R VEET) did not inhibit infection (open circles). (D) Inhibition of dengue viruses (DENV2, DENV3), but not H1N1 or H3N2 influenza viruses (IAVs), was observed using the VIVIT inhibitor (solid lines); scrambled VEET peptide in dashed lines. (E) Treatment of cells with VIVIT up to 6 h after infection by dengue immune complexes significantly inhibited infection. (F) Model for susceptibility to clinically significant dengue infections in infants. Infants of mothers who are dengue-immune and have ≥10% afucosylated anti-E IgGs are at elevated risk for symptomatic primary dengue infections due to modulation of FcγRIIIa-expressing cells during infection. (G) Model for the roles of FcγRIIa, FcγRIIIa, and afucosylated dengue immune complexes in ADE. While FcγRIIa supports a majority of viral attachment and entry, FcγRIIIa signaling increases cellular ITAM signaling. ITAM signaling, in turn, triggers calcium flux and modulates the calcineurin signaling network, which supports dengue virus replication. *p

    Journal: Cell reports

    Article Title: Maternal Anti-Dengue IgG Fucosylation Predicts Susceptibility to Dengue Disease in Infants

    doi: 10.1016/j.celrep.2020.107642

    Figure Lengend Snippet: Inhibition of Dengue Infection through Targeted Inhibition of ITAM Signaling Pathway Elements (A) A small molecule that targets Syk tyrosine kinase (Syk TK), PRT, inhibited dengue infection in FcγRNa + FcγRNIa + U937 monocytes. (B and C) A small-molecule inhibitor, INCA-6 (B), and a peptide inhibitor, 11R-VIVIT (VIVIT) (C), which act by blocking calcineurin-substrate interactions at the calcineurin PxIxIT site, inhibited dengue infections. A scrambled peptide containing the amino acids found in VIVIT (11R VEET) did not inhibit infection (open circles). (D) Inhibition of dengue viruses (DENV2, DENV3), but not H1N1 or H3N2 influenza viruses (IAVs), was observed using the VIVIT inhibitor (solid lines); scrambled VEET peptide in dashed lines. (E) Treatment of cells with VIVIT up to 6 h after infection by dengue immune complexes significantly inhibited infection. (F) Model for susceptibility to clinically significant dengue infections in infants. Infants of mothers who are dengue-immune and have ≥10% afucosylated anti-E IgGs are at elevated risk for symptomatic primary dengue infections due to modulation of FcγRIIIa-expressing cells during infection. (G) Model for the roles of FcγRIIa, FcγRIIIa, and afucosylated dengue immune complexes in ADE. While FcγRIIa supports a majority of viral attachment and entry, FcγRIIIa signaling increases cellular ITAM signaling. ITAM signaling, in turn, triggers calcium flux and modulates the calcineurin signaling network, which supports dengue virus replication. *p

    Article Snippet: For infections involving inhibitors or activators of ITAM signaling, U937s were treated with titrated inhibitors/activators: Ionomycin (Invivogen; inh-ion), PRT062607 (Fisher Scientific; NC0664362), INCA-6 (Abcam; ab145864), 11R-VIVIT (Thermo Fisher; CM102281.1), or 11R-VEET (Thermo Fisher; CM102281.2) in complete RPMI media for 1h-6h at 37C prior to, during or following infection as indicated.

    Techniques: Inhibition, Infection, Blocking Assay, Expressing

    Signal 1 determines resistance to ABT-737 in activated T cells. ( a ) Selective inhibitors of signal 1, 2 and 3 were added to an MLR during the stimulation phase with BM3.3 splenocytes reacting against CD8 T-cell-depleted B6 splenocytes to investigate the role of the different T-cell activation pathways for resistance to ABT-737(1 μ M, during additional 12 h of culture). The calcineurin inhibitor and signal 1 blocker CsA prevented resistance to ABT-737 in a concentration-dependent manner, whereas inhibition of signal 2 by CTLA4Ig and signal 3 by rapamycin did not influence this process. ( b ) The results obtained with CsA were confirmed by other inhibitors of this same pathway, namely the alternative calcineurin inhibitor tacrolimus and the NFAT inhibitor VIVIT-R. Cell viability of BM3.3 CD8 T cells was assessed by PI exclusion in FACS in at least three independent experiments. Percentage of cells treated with vehicle is given. Statistical comparison of ABT-737 versus vehicle: * P

    Journal: Cell Death & Disease

    Article Title: Resistance to ABT-737 in activated T lymphocytes: molecular mechanisms and reversibility by inhibition of the calcineurin-NFAT pathway

    doi: 10.1038/cddis.2012.38

    Figure Lengend Snippet: Signal 1 determines resistance to ABT-737 in activated T cells. ( a ) Selective inhibitors of signal 1, 2 and 3 were added to an MLR during the stimulation phase with BM3.3 splenocytes reacting against CD8 T-cell-depleted B6 splenocytes to investigate the role of the different T-cell activation pathways for resistance to ABT-737(1 μ M, during additional 12 h of culture). The calcineurin inhibitor and signal 1 blocker CsA prevented resistance to ABT-737 in a concentration-dependent manner, whereas inhibition of signal 2 by CTLA4Ig and signal 3 by rapamycin did not influence this process. ( b ) The results obtained with CsA were confirmed by other inhibitors of this same pathway, namely the alternative calcineurin inhibitor tacrolimus and the NFAT inhibitor VIVIT-R. Cell viability of BM3.3 CD8 T cells was assessed by PI exclusion in FACS in at least three independent experiments. Percentage of cells treated with vehicle is given. Statistical comparison of ABT-737 versus vehicle: * P

    Article Snippet: Antimycin A, CsA, rapamycin and tacrolimus were purchased from EnzoBiochem (Farmingdale, NY, USA), cycloheximide from Sigma-Aldrich (Buchs, Switzerland), VIVIT-R from Calbiochem (Merck, Darmstadt, Germany), obatoclax (GX15-070) from Selleck (Houston, TX, USA).

    Techniques: Activation Assay, Concentration Assay, Inhibition, FACS

    PLCδ1 silencing activates p38 MAPK. ( a ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs, and grown in medium either with (high Ca 2+ ) or without (low Ca 2+ ) 1.2 mM CaCl 2 . β -actin was used as a loading control. Results are representative of two trials. ( b ) Extracellular ATP concentrations were measured in conditioned medium of NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs. ( N =4 in each group). ( c ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in the presence of an NFAT inhibitor (11 R-VIVIT; treated for 24 h with the concentrations indicated). β -actin was used as a loading control. Results are representative of two trials. ( d , e ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in human organotypic skin cultures treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs ( d ) and the epidermis of control and keratinocyte-specific PLC δ 1 knockout (cKO) mice ( e ). β -actin was used as a loading control. Results are representative of two trials ( d ) or three animals per group ( e ). ( f ) Skin from control and PLC δ 1 cKO mice were stained with an antibody against phosphorylated-p38 MAPK (brown). Scale bar=50 μ m. Images are representative of three animals per group. Stained sections were assessed in a blinded fashion by two independent observers. ( g ) Immunoblotting of phosphorylated (p-HSP27) and total HSP27 in NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs, and either vehicle or a p38 MAPK inhibitor (SB202190). β -actin was used as a loading control. Results are representative of two trials. ( h ) NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs were pretreated with either vehicle or SB202190, and then exposed to 2 mM CaCl 2 for 15 min. RhoA-GTP levels were determined using G-LISA in NHEK stimulated with Ca 2+ . RhoA-GTP levels were normalized to the total protein amount. Data are represented as mean±S.E.M. ( N =3 in each group). ( i ) Immunofluorescence detection of ZO-1 in NHEK treated with either non- (control) or PLCδ1 -targeting siRNAs, grown in medium containing 1.2 mM CaCl 2 for 24 h in the presence of either vehicle or SB202190. Scale bar=50 μ m. Images are representative of three trials. ( j ) Skin samples of control and keratinocyte-specific PLC δ 1 knockout (cKO) mice treated with either vehicle or SB202190, and were stained with H E or an antibody against ZO-1. Dotted lines denote the skin surface. Scale bar=50 μ m (H E), and 20 μ m (ZO-1). Images are representative of five animals per group. ( k ) Lucifer yellow fluorescence was visualized in sections of human organotypic skin cultures treated with either non- (control) or PLCδ1 -targeting (PLC δ 1siRNA) siRNAs. Either vehicle or SB202190 was added to the medium in the last 72 h of culture. Nuclei were counter-stained with Hoechst (blue). Dotted lines denote the skin culture surface. Scale bar=30 μ m. Images are representative of three trials. Stained sections were assessed in a blinded fashion by two independent observers. ( a , c , d , e and g ) Relative ratios of phosphorylated to total protein (Phospho/Total) are denoted below each panel. Statistical significance was assessed using Welch's t -test. n.s; not significant

    Journal: Cell Death and Differentiation

    Article Title: Phospholipase Cδ1 regulates p38 MAPK activity and skin barrier integrity

    doi: 10.1038/cdd.2017.56

    Figure Lengend Snippet: PLCδ1 silencing activates p38 MAPK. ( a ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs, and grown in medium either with (high Ca 2+ ) or without (low Ca 2+ ) 1.2 mM CaCl 2 . β -actin was used as a loading control. Results are representative of two trials. ( b ) Extracellular ATP concentrations were measured in conditioned medium of NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs. ( N =4 in each group). ( c ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in the presence of an NFAT inhibitor (11 R-VIVIT; treated for 24 h with the concentrations indicated). β -actin was used as a loading control. Results are representative of two trials. ( d , e ) Immunoblotting of phosphorylated (p-p38) and total p38 MAPK in human organotypic skin cultures treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs ( d ) and the epidermis of control and keratinocyte-specific PLC δ 1 knockout (cKO) mice ( e ). β -actin was used as a loading control. Results are representative of two trials ( d ) or three animals per group ( e ). ( f ) Skin from control and PLC δ 1 cKO mice were stained with an antibody against phosphorylated-p38 MAPK (brown). Scale bar=50 μ m. Images are representative of three animals per group. Stained sections were assessed in a blinded fashion by two independent observers. ( g ) Immunoblotting of phosphorylated (p-HSP27) and total HSP27 in NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs, and either vehicle or a p38 MAPK inhibitor (SB202190). β -actin was used as a loading control. Results are representative of two trials. ( h ) NHEK treated with either non- (control) or PLCδ1 -targeting (siPLC δ 1#1 and siPLC δ 1#2) siRNAs were pretreated with either vehicle or SB202190, and then exposed to 2 mM CaCl 2 for 15 min. RhoA-GTP levels were determined using G-LISA in NHEK stimulated with Ca 2+ . RhoA-GTP levels were normalized to the total protein amount. Data are represented as mean±S.E.M. ( N =3 in each group). ( i ) Immunofluorescence detection of ZO-1 in NHEK treated with either non- (control) or PLCδ1 -targeting siRNAs, grown in medium containing 1.2 mM CaCl 2 for 24 h in the presence of either vehicle or SB202190. Scale bar=50 μ m. Images are representative of three trials. ( j ) Skin samples of control and keratinocyte-specific PLC δ 1 knockout (cKO) mice treated with either vehicle or SB202190, and were stained with H E or an antibody against ZO-1. Dotted lines denote the skin surface. Scale bar=50 μ m (H E), and 20 μ m (ZO-1). Images are representative of five animals per group. ( k ) Lucifer yellow fluorescence was visualized in sections of human organotypic skin cultures treated with either non- (control) or PLCδ1 -targeting (PLC δ 1siRNA) siRNAs. Either vehicle or SB202190 was added to the medium in the last 72 h of culture. Nuclei were counter-stained with Hoechst (blue). Dotted lines denote the skin culture surface. Scale bar=30 μ m. Images are representative of three trials. Stained sections were assessed in a blinded fashion by two independent observers. ( a , c , d , e and g ) Relative ratios of phosphorylated to total protein (Phospho/Total) are denoted below each panel. Statistical significance was assessed using Welch's t -test. n.s; not significant

    Article Snippet: Either 20 μ M SB202190 or 11 R-VIVIT (25 μ M; Millipore) was added to the assay medium for the last 48 or 72 h of culture.

    Techniques: Planar Chromatography, Knock-Out, Mouse Assay, Staining, Immunofluorescence, Fluorescence

    Anchored PKA and CaN have opposing effects on L-type Ca 2+ current in cultured hippocampal pyramidal neurons Peptides and other drugs were applied intracellularly via the patch pipet. Representative current recordings are shown for each time course as indicated; black records and colored records show currents 60 s (time point 1) and 250 s (time point 2) after establishing the whole cell recording configuration, respectively. All scale bars indicate 600 pA. ( A ) Average peak current time course during dialysis of intracellular recording solution alone (Con, ) or in the presence of either VIVIT ( ) or Ht31 ( ). ( B ) The effect of CsA dialysis ( ) and co-application of VIVIT and PKI ( ). ( C and D ) as in A and B, but with FSK added to the intracellular recording solution.

    Journal: Neuron

    Article Title: AKAP79/150 Anchoring of Calcineurin Controls Neuronal L-type Ca2+ Channel Activity and Nuclear Signaling

    doi: 10.1016/j.neuron.2007.06.032

    Figure Lengend Snippet: Anchored PKA and CaN have opposing effects on L-type Ca 2+ current in cultured hippocampal pyramidal neurons Peptides and other drugs were applied intracellularly via the patch pipet. Representative current recordings are shown for each time course as indicated; black records and colored records show currents 60 s (time point 1) and 250 s (time point 2) after establishing the whole cell recording configuration, respectively. All scale bars indicate 600 pA. ( A ) Average peak current time course during dialysis of intracellular recording solution alone (Con, ) or in the presence of either VIVIT ( ) or Ht31 ( ). ( B ) The effect of CsA dialysis ( ) and co-application of VIVIT and PKI ( ). ( C and D ) as in A and B, but with FSK added to the intracellular recording solution.

    Article Snippet: Cyclosporin A, forskolin and PKI were obtained from Sigma; ω-CTx-GVIA, ω-CTx-MVIIC and myr-PKI were obtained from EMD Biosciences; CaN457-482 was obtained from Biomol; St -Ht31 and St-Ht31pro were obtained from Promega; and St-VIVIT was synthesized by Sigma-Genosys.

    Techniques: Cell Culture

    The mechanism of SLC4A2 in wear particle-induced osteoclast differentiation and function. (A) 11R-VIVIT, an inhibitor of NFATc1 suppressed up-regulation of Slc4a2 expression during wear particle-induced osteoclastogenesis. (B) Expression of Nfatc1 was inhibited by knockdown of Slc4a2 with shRNA3 unexpectedly. (C–E) Suppression of osteoclast-specific gene expression ( Trap , Ctsk , Ctr ) with knockdown of Slc4a2 with shRNA3. (F) Knockdown of Slc4a2 , using shRNA3, inhibits F-actin ring formation in vitro . (G) The size of F-actin rings, measured using ImageJ ( ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Targeting Anion Exchange of Osteoclast, a New Strategy for Preventing Wear Particles Induced- Osteolysis

    doi: 10.3389/fphar.2018.01291

    Figure Lengend Snippet: The mechanism of SLC4A2 in wear particle-induced osteoclast differentiation and function. (A) 11R-VIVIT, an inhibitor of NFATc1 suppressed up-regulation of Slc4a2 expression during wear particle-induced osteoclastogenesis. (B) Expression of Nfatc1 was inhibited by knockdown of Slc4a2 with shRNA3 unexpectedly. (C–E) Suppression of osteoclast-specific gene expression ( Trap , Ctsk , Ctr ) with knockdown of Slc4a2 with shRNA3. (F) Knockdown of Slc4a2 , using shRNA3, inhibits F-actin ring formation in vitro . (G) The size of F-actin rings, measured using ImageJ ( ∗ P

    Article Snippet: The 11R-VIVIT peptide (RRRRRRRRRRR-GGG-MAGPHPVIVITGPHEE) was obtained from Sigma Genosys (Woodlands, TX, United States).

    Techniques: Expressing, In Vitro

    Effects of intrathecal treatment with 11R-VIVIT on the expression levels of NFATc4, CCR2, and BK α 1 in the DRG of nerve-injured rats. 11R-VIVIT (20 μ g, twice/day, n = 8) or saline ( n = 8) was injected for the first 5 days after nerve injury. The mRNA levels of NFATc4 (A), CCR2 (B), and BK α 1 (C) were quantified using real-time PCR from left L5 and L6 DRG tissues obtained 14 days after nerve injury ( n = 8 in each group). * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Upregulation of Nuclear Factor of Activated T-Cells by Nerve Injury Contributes to Development of Neuropathic Pain

    doi: 10.1124/jpet.112.202192

    Figure Lengend Snippet: Effects of intrathecal treatment with 11R-VIVIT on the expression levels of NFATc4, CCR2, and BK α 1 in the DRG of nerve-injured rats. 11R-VIVIT (20 μ g, twice/day, n = 8) or saline ( n = 8) was injected for the first 5 days after nerve injury. The mRNA levels of NFATc4 (A), CCR2 (B), and BK α 1 (C) were quantified using real-time PCR from left L5 and L6 DRG tissues obtained 14 days after nerve injury ( n = 8 in each group). * P

    Article Snippet: The NFATc inhibitor 11R-VIVIT (Calbiochem, Darmstadt, Germany) was dissolved in sterile saline to 4 mg/ml.

    Techniques: Expressing, Injection, Real-time Polymerase Chain Reaction

    Effect of intrathecal injection of 11R-VIVIT or FK-506 on the development of pain hypersensitivity after nerve injury. (A) Time course of the development of tactile allodynia in vehicle- and 11R-VIVIT–treated rats ( n = 8 rats in each group). (B) Time course of the development of tactile allodynia in vehicle- and FK-506–treated rats ( n = 11 rats in each group). Tactile allodynia was tested using von Frey filaments. Rats were treated with 11R-VIVIT or FK-506 for the first 5 days after L5 and L6 spinal nerve ligation. * P

    Journal: The Journal of Pharmacology and Experimental Therapeutics

    Article Title: Upregulation of Nuclear Factor of Activated T-Cells by Nerve Injury Contributes to Development of Neuropathic Pain

    doi: 10.1124/jpet.112.202192

    Figure Lengend Snippet: Effect of intrathecal injection of 11R-VIVIT or FK-506 on the development of pain hypersensitivity after nerve injury. (A) Time course of the development of tactile allodynia in vehicle- and 11R-VIVIT–treated rats ( n = 8 rats in each group). (B) Time course of the development of tactile allodynia in vehicle- and FK-506–treated rats ( n = 11 rats in each group). Tactile allodynia was tested using von Frey filaments. Rats were treated with 11R-VIVIT or FK-506 for the first 5 days after L5 and L6 spinal nerve ligation. * P

    Article Snippet: The NFATc inhibitor 11R-VIVIT (Calbiochem, Darmstadt, Germany) was dissolved in sterile saline to 4 mg/ml.

    Techniques: Injection, Ligation

    NFATs are relevant in human oligodendrocytes. a NFAT expression in human iOLs (grey bars) and NPCs (open bars) by qrtPCR after normalization to GAPDH . Average NFAT levels in NPCs were set to 1 ( NFATc1 : 1.4 ± 0.3; NFATc2 : 14.6 ± 1.9; NFATc3 : 1.6 ± 0.3 and NFATc4 : 0.9 ± 0.2). b Immunocytochemistry of MBP-positive (red) iOL with antibodies directed against NFATs (white in upper row, green in lower row); counterstaining with Hoechst dye (blue). c – g Analysis of differentiating iOL cultured 21 ( c , e , h ) or 35 days ( d , f , g , i , j ) in absence (Ctr, open bars) or presence of 0.1 (light grey bars) to 0.5 µM (dark grey bars) VIVIT ( c – g , n = 4) or 1 (light grey bars) to 10 µM (dark grey bars) FK506 ( h – j , n = 3–4). VIVIT/FK506 incubation was continuous ( c – f , h , i ) or from days 21–35 ( g , j ). Cultures were stained with anti-O4 (green, c ) and anti-MBP antibodies (green, d ). From staining and FACS analysis (see Suppl. Fig. 7c, d ) the fraction of O4-positive cells after 21 days ( e , h ) and MBP-positive cells after 35 days ( f , i ) was determined. For treatment during days 21–35, the fraction of FACS-sorted O4-positive cells was determined that had reached a MBP-positive stage ( g , j ). The relative number of marker-positive cells under control conditions was set to 1 and used to normalize in pairwise fashion (values: 1 for control, 0.97 ± 0.01 for 21 days 0.1 µM VIVIT, 0.68 ± 0.06 for 21 days 0.5 µM VIVIT, 0.86 ± 0.10 for 35 days 0.1 µM VIVIT, 0.60 ± 0.10 for 35 days 0.5 µM VIVIT, 0.82 ± 0.05 for 0.1 µM VIVIT from days 21–35, 0.86 ± 0.04 for 0.5 µM VIVIT from days 21–35, 0.99 ± 0.21 for 21 days 1 µM FK506, 0.39 ± 0.17 for 21 days 10 µM FK506, 1.12 ± 0.36 for 35 days 1 µM FK506, 0.55 ± 0.09 for 35 days 10 µM FK506, 0.83 ± 0.17 for 1 µM FK506 from days 21–35 and 0.86 ± 0.10 for 10 µM FK506 from days 21–35). k – n NFAT immunohistochemistry in human brain tissue. Antibodies directed against NFATc3 and NFATc4 (red in k and l , black in m ) were combined with anti-NOGOA antibodies (green in k and l , red in m ). Arrows mark double positive cells, arrowheads NOGOA-positive oligodendrocytes without NFATc4. n Percentage of NOGOA-positive oligodendrocytes with nuclear NFATc4 in periplaque white matter (86 ± 4%), active and demyelinating lesions (41 ± 8%), active and post-demyelinating lesions (41 ± 8%), active and remyelinating plaques (59 ± 19%), and control CNS tissue (99 ± 1%). o Model of oligodendroglial Nfat action. Statistical significance was determined by two-tailed Student’s t -test ( a ) or Bonferroni-corrected one-way ANOVA ( e – j , n ) (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). Scale bars, 50 µm

    Journal: Nature Communications

    Article Title: Nfat/calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning

    doi: 10.1038/s41467-018-03336-3

    Figure Lengend Snippet: NFATs are relevant in human oligodendrocytes. a NFAT expression in human iOLs (grey bars) and NPCs (open bars) by qrtPCR after normalization to GAPDH . Average NFAT levels in NPCs were set to 1 ( NFATc1 : 1.4 ± 0.3; NFATc2 : 14.6 ± 1.9; NFATc3 : 1.6 ± 0.3 and NFATc4 : 0.9 ± 0.2). b Immunocytochemistry of MBP-positive (red) iOL with antibodies directed against NFATs (white in upper row, green in lower row); counterstaining with Hoechst dye (blue). c – g Analysis of differentiating iOL cultured 21 ( c , e , h ) or 35 days ( d , f , g , i , j ) in absence (Ctr, open bars) or presence of 0.1 (light grey bars) to 0.5 µM (dark grey bars) VIVIT ( c – g , n = 4) or 1 (light grey bars) to 10 µM (dark grey bars) FK506 ( h – j , n = 3–4). VIVIT/FK506 incubation was continuous ( c – f , h , i ) or from days 21–35 ( g , j ). Cultures were stained with anti-O4 (green, c ) and anti-MBP antibodies (green, d ). From staining and FACS analysis (see Suppl. Fig. 7c, d ) the fraction of O4-positive cells after 21 days ( e , h ) and MBP-positive cells after 35 days ( f , i ) was determined. For treatment during days 21–35, the fraction of FACS-sorted O4-positive cells was determined that had reached a MBP-positive stage ( g , j ). The relative number of marker-positive cells under control conditions was set to 1 and used to normalize in pairwise fashion (values: 1 for control, 0.97 ± 0.01 for 21 days 0.1 µM VIVIT, 0.68 ± 0.06 for 21 days 0.5 µM VIVIT, 0.86 ± 0.10 for 35 days 0.1 µM VIVIT, 0.60 ± 0.10 for 35 days 0.5 µM VIVIT, 0.82 ± 0.05 for 0.1 µM VIVIT from days 21–35, 0.86 ± 0.04 for 0.5 µM VIVIT from days 21–35, 0.99 ± 0.21 for 21 days 1 µM FK506, 0.39 ± 0.17 for 21 days 10 µM FK506, 1.12 ± 0.36 for 35 days 1 µM FK506, 0.55 ± 0.09 for 35 days 10 µM FK506, 0.83 ± 0.17 for 1 µM FK506 from days 21–35 and 0.86 ± 0.10 for 10 µM FK506 from days 21–35). k – n NFAT immunohistochemistry in human brain tissue. Antibodies directed against NFATc3 and NFATc4 (red in k and l , black in m ) were combined with anti-NOGOA antibodies (green in k and l , red in m ). Arrows mark double positive cells, arrowheads NOGOA-positive oligodendrocytes without NFATc4. n Percentage of NOGOA-positive oligodendrocytes with nuclear NFATc4 in periplaque white matter (86 ± 4%), active and demyelinating lesions (41 ± 8%), active and post-demyelinating lesions (41 ± 8%), active and remyelinating plaques (59 ± 19%), and control CNS tissue (99 ± 1%). o Model of oligodendroglial Nfat action. Statistical significance was determined by two-tailed Student’s t -test ( a ) or Bonferroni-corrected one-way ANOVA ( e – j , n ) (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001). Scale bars, 50 µm

    Article Snippet: Where indicated, rodent or human oligodendroglial cultures were incubated with 0.1–5 µM of the NFAT inhibitor 11R-VIVIT (Merck Millipore), 0.1–10 µM of the calcineurin inhibitor FK506 (Bertin Pharma), 0.5–1 µM ionomycin (Cell Signaling) or 0.05–0.1% DMSO as solvent control over different time periods.

    Techniques: Expressing, Immunocytochemistry, Cell Culture, Incubation, Staining, FACS, Marker, Immunohistochemistry, Two Tailed Test

    Nfat/calcineurin signaling is required for oligodendroglial differentiation in culture. a – c Analysis of myelin gene expression in primary mouse oligodendroglial cells cultured for 48 h under differentiating conditions in the absence (Ctr, open bar) or presence of 1 µM VIVIT (VIVIT, grey bars). Incubation with VIVIT was restricted to the first 24 h (light grey bars) or second 24 h (grey bars) of incubation or throughout the whole cultivation period (dark grey bars). Cultures were stained with antibodies directed against Mbp (red) and counterstained with Hoechst (blue). Scale bar, 50 µm ( a ). From immunocytochemical stainings the fraction of Mbp-positive cells was determined ( b ) ( n = 3). The relative number of Mbp-positive cells present under control conditions was arbitrarily set to 1 and used to normalize in pairwise fashion (values: 1 for control conditions, 0.37 ± 0.11 for VIVIT treatment during the first 24 h, 0.75 ± 0.09 for VIVIT treatment during the second 24 h, 0.23 ± 0.11 for 48 h VIVIT treatment). RNA from these cultures was also used to perform qrtPCR and determine Mbp levels ( c ) ( n = 3). The amount of Mbp transcripts present after 48 h under control conditions was arbitrarily set to 1 and used to normalize (values: 1 for control conditions, 0.23 ± 0.10 for VIVIT treatment during the first 24 h, 0.48 ± 0.06 for VIVIT treatment during the second 24 h, 0.10 ± 0.02 for 48 h VIVIT treatment). d – f Analysis of myelination in cerebellar slices of newborn mice after 12 days of culture in the absence (Ctr, open bars) and presence (VIVIT, grey bars) of 1 µM VIVIT ( n = 3). Cultures were stained ( d ) with antibodies directed against Mbp (red) and Neurofilament L (Nefl, green) and counterstained with DAPI (blue). Scale bar, 20 µm. The extent of myelination was assessed by determining the Nefl-positive area co-stained with Mbp ( e ). The Mbp/Nefl ratio under control conditions was arbitrarily set to 1 and used to normalize in pairwise fashion (values: 1 for control conditions, 0.72 ± 0.09 for VIVIT treatment). RNA prepared from these cultures was used to determine Mbp and Nefl expression ( f ). The amount of Mbp relative to Nefl transcripts under control conditions was arbitrarily set to 1 (values: 1 for control conditions, 0.71 ± 0.08 for VIVIT treatment). Statistical significance was determined by Bonferroni-corrected one-way ANOVA in ( b ) and ( c ) and two-tailed Student’s t -test in ( e ) and ( f ) (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001)

    Journal: Nature Communications

    Article Title: Nfat/calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning

    doi: 10.1038/s41467-018-03336-3

    Figure Lengend Snippet: Nfat/calcineurin signaling is required for oligodendroglial differentiation in culture. a – c Analysis of myelin gene expression in primary mouse oligodendroglial cells cultured for 48 h under differentiating conditions in the absence (Ctr, open bar) or presence of 1 µM VIVIT (VIVIT, grey bars). Incubation with VIVIT was restricted to the first 24 h (light grey bars) or second 24 h (grey bars) of incubation or throughout the whole cultivation period (dark grey bars). Cultures were stained with antibodies directed against Mbp (red) and counterstained with Hoechst (blue). Scale bar, 50 µm ( a ). From immunocytochemical stainings the fraction of Mbp-positive cells was determined ( b ) ( n = 3). The relative number of Mbp-positive cells present under control conditions was arbitrarily set to 1 and used to normalize in pairwise fashion (values: 1 for control conditions, 0.37 ± 0.11 for VIVIT treatment during the first 24 h, 0.75 ± 0.09 for VIVIT treatment during the second 24 h, 0.23 ± 0.11 for 48 h VIVIT treatment). RNA from these cultures was also used to perform qrtPCR and determine Mbp levels ( c ) ( n = 3). The amount of Mbp transcripts present after 48 h under control conditions was arbitrarily set to 1 and used to normalize (values: 1 for control conditions, 0.23 ± 0.10 for VIVIT treatment during the first 24 h, 0.48 ± 0.06 for VIVIT treatment during the second 24 h, 0.10 ± 0.02 for 48 h VIVIT treatment). d – f Analysis of myelination in cerebellar slices of newborn mice after 12 days of culture in the absence (Ctr, open bars) and presence (VIVIT, grey bars) of 1 µM VIVIT ( n = 3). Cultures were stained ( d ) with antibodies directed against Mbp (red) and Neurofilament L (Nefl, green) and counterstained with DAPI (blue). Scale bar, 20 µm. The extent of myelination was assessed by determining the Nefl-positive area co-stained with Mbp ( e ). The Mbp/Nefl ratio under control conditions was arbitrarily set to 1 and used to normalize in pairwise fashion (values: 1 for control conditions, 0.72 ± 0.09 for VIVIT treatment). RNA prepared from these cultures was used to determine Mbp and Nefl expression ( f ). The amount of Mbp relative to Nefl transcripts under control conditions was arbitrarily set to 1 (values: 1 for control conditions, 0.71 ± 0.08 for VIVIT treatment). Statistical significance was determined by Bonferroni-corrected one-way ANOVA in ( b ) and ( c ) and two-tailed Student’s t -test in ( e ) and ( f ) (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001)

    Article Snippet: Where indicated, rodent or human oligodendroglial cultures were incubated with 0.1–5 µM of the NFAT inhibitor 11R-VIVIT (Merck Millipore), 0.1–10 µM of the calcineurin inhibitor FK506 (Bertin Pharma), 0.5–1 µM ionomycin (Cell Signaling) or 0.05–0.1% DMSO as solvent control over different time periods.

    Techniques: Expressing, Cell Culture, Incubation, Staining, Mouse Assay, Two Tailed Test

    Sox10 activates oligodendroglial Nkx2.2 expression via ECR19. a QrtPCR analysis of Nkx2.2 expression in mouse oligodendroglial cultures after 6 and 24 h differentiation in absence (Ctr, open bars) or presence of 1 µM VIVIT (grey bars) ( n = 3). Nkx2.2 amounts under control conditions after 6 h were set to 1 (values: 1 ± 0.20 for Ctr and 0.65 ± 0.14 for VIVIT after 6 h, 3.21 ± 0.12 for Ctr and 1.92 ± 0.25 for VIVIT after 24 h). b Rat Nkx2.2 locus with exons (solid pink boxes), ChIP-Seq peaks (GEO accession numbers GSE64703 and GSE42447) for Olig2 (green boxes) and Sox10 (blue boxes) in ECRs (open pink boxes) at −115, −19, +5, and +45 kilobases relative to the transcriptional start site (arrow) and ChIP control regions (Ctr1 and Ctr2, grey boxes). c N2a cell transfections with luciferase reporters carrying Nkx2.2 ECRs in absence (−) or presence of low (+, light grey bars) and high Sox10 amounts (++, dark grey bars) ( n = 3). Sox10-dependent fold inductions ± SEM were determined after 48 h with activities in the absence of Sox10 set to 1 (ECR115: 1.6 ± 0.3 at both concentrations; ECR19: 32.9 ± 7.1 and 198.3 ± 26.0; ECR5: 44.6 ± 16.4 and 107.3 ± 10.7; ECR45: 4.5 ± 2.1 and 10.6 ± 0.9). d ChIP on differentiating rat oligodendrocytes after 4 days ( n = 3) using rabbit pre-immune (pre IS, open bars) and anti-Sox10 (αSox10, grey bars) antiserum. Amounts of immunoprecipitated ECR19 and control region (Ctr1) were qPCR-determined and are presented as percent of input (ECR19: 0.012 ± 0.004 for pre IS and 0.073 ± 0.013 for αSox10; Ctr1: 0.016 ± 0.005 for pre IS and 0.021 ± 0.005 for αSox10). e Localization of binding sites for Sox10 (putative, light blue; EMSA-confirmed, dark blue), Nfat (brown) and Olig2 (green) in ECR19. Numbers on left and right correspond to mouse ECR19 positions relative to transcriptional start site. For EMSA and sequences, see Suppl. Fig. 4c, d . f N2a cell transfections with wildtype (WT) or mutant ECR19 luciferase reporters with inactivated Sox10 binding sites (S4m, S7/7am, and S4/7/7am) in absence (−) or presence (+, grey bars) of Sox10 ( n = 3). Sox10-dependent fold inductions ± SEM were determined 48 h post transfection (WT: 106.7 ± 0.7; S4m: 54.6 ± 9.1; S7/7am 23.4 ± 0.9; S4/7/7am 14.6 ± 2.3). g Sox10 immunohistochemistry (green) on cortical slices 7 days after transduction with retroviruses carrying tdTomato reporters (red) under control of a minimal promoter (control) or minimal promoter and ECR19 (ECR19). Co-expression is in yellow, DAPI counterstain in blue. Scale bar, 50 µm. Statistical significance was determined by two-tailed Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001)

    Journal: Nature Communications

    Article Title: Nfat/calcineurin signaling promotes oligodendrocyte differentiation and myelination by transcription factor network tuning

    doi: 10.1038/s41467-018-03336-3

    Figure Lengend Snippet: Sox10 activates oligodendroglial Nkx2.2 expression via ECR19. a QrtPCR analysis of Nkx2.2 expression in mouse oligodendroglial cultures after 6 and 24 h differentiation in absence (Ctr, open bars) or presence of 1 µM VIVIT (grey bars) ( n = 3). Nkx2.2 amounts under control conditions after 6 h were set to 1 (values: 1 ± 0.20 for Ctr and 0.65 ± 0.14 for VIVIT after 6 h, 3.21 ± 0.12 for Ctr and 1.92 ± 0.25 for VIVIT after 24 h). b Rat Nkx2.2 locus with exons (solid pink boxes), ChIP-Seq peaks (GEO accession numbers GSE64703 and GSE42447) for Olig2 (green boxes) and Sox10 (blue boxes) in ECRs (open pink boxes) at −115, −19, +5, and +45 kilobases relative to the transcriptional start site (arrow) and ChIP control regions (Ctr1 and Ctr2, grey boxes). c N2a cell transfections with luciferase reporters carrying Nkx2.2 ECRs in absence (−) or presence of low (+, light grey bars) and high Sox10 amounts (++, dark grey bars) ( n = 3). Sox10-dependent fold inductions ± SEM were determined after 48 h with activities in the absence of Sox10 set to 1 (ECR115: 1.6 ± 0.3 at both concentrations; ECR19: 32.9 ± 7.1 and 198.3 ± 26.0; ECR5: 44.6 ± 16.4 and 107.3 ± 10.7; ECR45: 4.5 ± 2.1 and 10.6 ± 0.9). d ChIP on differentiating rat oligodendrocytes after 4 days ( n = 3) using rabbit pre-immune (pre IS, open bars) and anti-Sox10 (αSox10, grey bars) antiserum. Amounts of immunoprecipitated ECR19 and control region (Ctr1) were qPCR-determined and are presented as percent of input (ECR19: 0.012 ± 0.004 for pre IS and 0.073 ± 0.013 for αSox10; Ctr1: 0.016 ± 0.005 for pre IS and 0.021 ± 0.005 for αSox10). e Localization of binding sites for Sox10 (putative, light blue; EMSA-confirmed, dark blue), Nfat (brown) and Olig2 (green) in ECR19. Numbers on left and right correspond to mouse ECR19 positions relative to transcriptional start site. For EMSA and sequences, see Suppl. Fig. 4c, d . f N2a cell transfections with wildtype (WT) or mutant ECR19 luciferase reporters with inactivated Sox10 binding sites (S4m, S7/7am, and S4/7/7am) in absence (−) or presence (+, grey bars) of Sox10 ( n = 3). Sox10-dependent fold inductions ± SEM were determined 48 h post transfection (WT: 106.7 ± 0.7; S4m: 54.6 ± 9.1; S7/7am 23.4 ± 0.9; S4/7/7am 14.6 ± 2.3). g Sox10 immunohistochemistry (green) on cortical slices 7 days after transduction with retroviruses carrying tdTomato reporters (red) under control of a minimal promoter (control) or minimal promoter and ECR19 (ECR19). Co-expression is in yellow, DAPI counterstain in blue. Scale bar, 50 µm. Statistical significance was determined by two-tailed Student’s t-test (* P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001)

    Article Snippet: Where indicated, rodent or human oligodendroglial cultures were incubated with 0.1–5 µM of the NFAT inhibitor 11R-VIVIT (Merck Millipore), 0.1–10 µM of the calcineurin inhibitor FK506 (Bertin Pharma), 0.5–1 µM ionomycin (Cell Signaling) or 0.05–0.1% DMSO as solvent control over different time periods.

    Techniques: Expressing, Chromatin Immunoprecipitation, Transfection, Luciferase, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Mutagenesis, Immunohistochemistry, Transduction, Two Tailed Test