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  • 99
    Millipore ionomycin
    Miro1-R272Q impairs cytosolic calcium handling and increases sensitivity to calcium stress . ( A ) iPSC-derived neurons were loaded with the cytosolic calcium indicator Fluo-4 AM for live-cell imaging. During imaging, cells were treated with 5 μM <t>ionomycin</t> in order to increase cytosolic calcium levels. Imaging was continued for 10 min with a 2 s interval. Images were obtained with a 40× objective. ( B ) Analysis of mean Fluo-4 AM fluorescence intensity (F1/F0) from images shown in A. Data indicated as mean ± SEM. Significance of first time points after treatment calculated by Holm–Sidak multiple t test; * P
    Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 46811 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher ionomycin
    Increased Tregs frequency in peripheral blood of CLL patients. a Gating strategy used to identify Tregs as CD4 + CD25 high FoxP3 + . Representative box plots of b Tregs frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15); c IL-10-secreting Tregs frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15); d Tregs subsets frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15), all after in vitro priming with IL-6 and phorbol 12-myristate 13-acetate (P), <t>ionomycin</t> (I) and monensin (M). All results are expressed as median and interquartile range. P value shown is obtained from the comparison between the indicated groups by exact non-parametric Mann–Whitney U test (*P
    Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4599 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Becton Dickinson pma ionomycin
    Curcumin suppresses CD69 expression in activated T cells. A and B , curcumin suppresses CD69 expression in T cells. Jurkat ( A ) and freshly isolated human peripheral blood T cells ( B ) were stimulated with <t>PMA</t> (5 ng/ml) and <t>ionomycin</t> (1 μ m ) in the
    Pma Ionomycin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 93/100, based on 847 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pma ionomycin
    Itk signaling is critical for regulating the differentiation and effector function of CD4 + T cells during EAE. A , B , Splenocytes isolated from day 10, day 17, and day 31 immunized WT and Itk −/− mice were stimulated with 5 μg/ml MOG peptide for 72 h. The cells were then restimulated with 10 μg/ml MOG peptide in the presence of Brefeldin A for 5 h, and CD4 + T cells were analyzed for the expression of IFNγ and IL-17A by FACS. Representative flow plots from day 10 is shown in A . B , Cytokine-producing cells quantified for IFNγ (left) or IL-17A (right) stimulation index (fold change in percentage of IFNγ or IL-17A-producing cells in response to MOG peptide compared with media controls). C , Cell culture supernatants of cells treated as in A were analyzed for the levels of IFNγ and IL-17A protein by ELISA. D , Splenocytes from A were stimulated with <t>PMA/ionomycin</t> (50 ng/1 μg/ml; Sigma) in the presence of Brefeldin A for 5 h and quantified for IFNγ (left) or IL-17A (right) stimulation index (fold change in percentage of IFNγ or IL-17A-producing cells in response to MOG peptide compared with media controls). E , Splenocytes obtained from day 17 immunized WT and Itk −/− mice were loaded with CFSE and stimulated with 5 μg/ml MOG peptide for 72 h. Alternatively H 3 -thymidine was added during the last 18 h of culture. CFSE-labeled cells were analyzed for proliferation of CD4 + T cells by FACS (top row), and data are represented as percentage CFSE diluted or fold increase in thymidine incorporation over media control (bottom row). Values are means ± SEMs, * p
    Pma Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1006 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore ionomycin calcium salt
    Idelalisib impairs calcium flux and TREM-1 signaling in neutrophils in vitro . PMN from healthy human donors were isolated, preincubated with idelalisib 1 µg/ml and loaded with FLUO-3/AM (both at 37 °C for 30 min) for calcium flux assay. ( A – C ) Fluorescence signals were detected by flow cytometry for 30 s. For PMN activation indicated stimuli (( A ) anti-TREM-1, ( B ) isotype matched control mAb, ( C ) <t>ionomycin)</t> was added. For cell stimulation via TREM-1 receptor (and isotype control) cross-linking was performed with a secondary antibody again after 30 s; fluorescence signals were gained for further 5 min. Ionomycin served as positive control. One out of three experiments is depicted. ( D ) For protein analysis PMN were activated with indicated stimuli (anti-TREM-1 antibody, matched control mAb, LPS) for 30 min after preincubated with idelalisib as described above. Proteins were extracted with an urea-based lysis buffer, SDS PAGE was performed and proteins were blotted by a semi-dry process. Protein activation was analyzed by staining of phosphorylated and non-phosphorylated proteins. ß-actin served as loading control. One out of three experiments is depicted.
    Ionomycin Calcium Salt, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson ionomycin
    Itch and WWP2 collaboratively regulate T H 2 differentiation. a , Frequency of splenic memory-like (CD44 hi CD62L − ) CD4 + T cells (left) and naive (CD25 − CD44 − CD62L hi ) CD4 + T cells (middle) from 6- to 7-week-old wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre and DKO mice (key; n = 8 per group), and flow cytometry of splenic naive CD4 + T cells from such mice, showing gating used (right). Numbers adjacent to outlined areas (right) indicate percent CD4 + CD25 − CD44 − CD62L hi cells. b , Gene-expression profiles (left) of splenic CD4 + T cells stimulated for 4h with PMA and <t>ionomycin,</t> showing genes expressed differentially (mean of normalized counts) in Wwp2 −/− , Itch f/f Cd4 -Cre or DKO cells (above plots) relative to their expression in wild-type cells, and expression of genes encoding selected cytokines (middle) or transcriptional regulators (right) in cells as at left (above plots), presented as log 2 reads per kilobase of exon per million mapped reads (RPKM) values. c , Frequency of CD4 + T cells with intracellular staining of IL-4 (left) or IFN-γ (right) among splenocytes obtained from mice as in a (key; n = 5–6 per group (IL-4) or n = 4–5 per group (IFN-γ)) and stimulated for 5h in vitro with PMA and ionomycin in the presence of the protein-transport inhibitor GolgiStop. d , ELISA of IL-4 (left) or IFN-γ (right) in supernatants of CD4 + T cells sorted from mice as in a (key; n = 3 per group) and stimulated for 48h in vitro with anti-CD3 plus anti-CD28. e , f , Immunoblot analysis of GATA-3 ( e ) and phosphorylated (p-) and total STAT6 ( e ) or STAT5 ( f ), as well as actin (loading control) (left margin), in lysates of CD4 + T cells sorted from mice as in a (above lanes) and left unstimulated (left) or stimulated for 16h with anti-CD3 plus anti-CD28 (right; α-CD3 + α-CD28); numbers below lanes indicate the ratio of phosphorylated protein to total protein. Each symbol ( a , c , d ) represents an individual mouse; small horizontal lines ( a ) indicate the mean (±s.d.). * P
    Ionomycin, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 8343 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Enzo Biochem ionomycin
    Effect of PKC and Ca 2+ and c-Src on Pyk2 Activation Acini were stimulated for 5 min with Cch (10 −4 M), PMA (10 −6 M), Cch and 2 mM EGTA, and <t>ionomycin</t> (10 −7 M). The amount of phosphorylated Pyk2 (Tyr 402 )/total Pyk2 is shown in A . Data are mean ± SEM from 3–6 independent experiments. Acini were also preincubated with PP1 for 15 min prior to stimulation with Cch. The amount of phosphorylated Pyk (Tyr 402 ) is shown in B . Data are mean ± SEM from 4 independent experiments. * indicates statistical significance from basal. # denotes statistical significance from Cch alone.
    Ionomycin, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 93/100, based on 770 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cayman Chemical ionomycin
    TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or <t>Ionomycin</t> ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).
    Ionomycin, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 93/100, based on 184 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Thermo Fisher pma ionomycin
    Characterization of the lymphoid compartment in PbA-infected B6, BXH2, [BXH2×B6]F1 and Irf1 −/− mice. Characterization of the lymphoid compartment was carried out as described in the legend of Figure 6 . Splenocytes were stained with CD45, TCRβ, CD4 and CD8 antibodies and representative cellular profiles are shown for each strain (A), where numbers indicate mean ± SD (gated as percentages of CD45 + cells). Absolute numbers are indicated in dot plots for total spleen CD4 + cells (B) and CD8 + cells (C). (D) Serum IFNγ levels were significantly lower in ECM-resistant BXH2, [BXH2×B6]F1 and Irf1 −/− mice. (E) IFNγ production was assayed in vitro in culture supernatants from infected mice with or without stimulation with <t>PMA/Ionomycin</t> or with IL12p70. The p-values were calculated relative to B6 controls with Student's t-test (**p
    Pma Ionomycin, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 735 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Merck & Co ionomycin
    TH2849 potentially induces autophagy by forming a complex with FKBP12 without interfering to the calcineurin/NFAT and IL2/p34cdc2/cyclin A signal pathway. (A) PC12 cells were pretreated with siRNA against FKBP12 for 48 h, and then, the protein and mRNA levels were examined. Representative images (B) and quantification (C) of PC12 cells with EGFP‐LC3 vesicles (autophagosomes) in the presence or absence of siRNA against FKBP12. PC12 cells co‐transfected with siRNA against FKBP12 and EGFP‐LC3 were treated with 1‰ DMSO, 1 μmol/L rapamycin, 10 μmol/L FK506, 1 μmol/L TH 2451 and 1 μmol/L TH 2849 for 24 h. (D) Representative images of distribution of EGFP‐NFATc1. The EGFP‐NFATc1 transfected MCF‐7 cells were treated with 2 μmol/L <t>ionomycin</t> or co‐treated with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263 and TH2287 for 2 h as indicated. (E ) Quantification of nucleus translocated EGFP‐NFATc1 cells in (D). (F) Representative Western blotting image of p34 cdc2 and cyclin A levels. Factor‐deprived CTLL‐2 cells were cultured for 14 h in basic medium only, 50 units/mL IL‐2 with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263, and TH2287 were added respectively during the last 1 hour. Quantification of relative p34 cdc2 (G) and cyclin A (H) expression levels in (F). All the substances were dissolved in DMSO. Scale bars: 10 μm. Data are shown as the mean ± SEM * P
    Ionomycin, supplied by Merck & Co, used in various techniques. Bioz Stars score: 92/100, based on 201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore calcium ionophore ionomycin
    Action of <t>ionomycin</t> on a csp mutant preparation. In the top ( 21°C ), mejps ( first two traces ) and the ejp are shown in a csp mutant preparation at permissive temperature. The characteristic failure of the ejp, but not the mejps, is illustrated in
    Calcium Ionophore Ionomycin, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 185 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Merck KGaA ionomycin
    Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore <t>ionomycin</t> was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.
    Ionomycin, supplied by Merck KGaA, used in various techniques. Bioz Stars score: 93/100, based on 174 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Abcam ionomycin
    Ca 2+ -evoked nNOS- and eNOS-derived NO generation profiles in <t>ionomycin</t> permeabilized cells (A) Mean curves represent NO generation profiles derived from eNOS (blue curve) and nNOS (green curve) in response to Ca 2+ addition. Experiments were performed in the presence of 5 μM ionomycin. NO recordings were performed using C-geNOp. (B) Bars represent respective statistics of panel A, maximum initial slope of nNOS- (green bar, n = 34/3) and eNOS-derived (blue bar, n = 36/3) NO signals. Mean values are shown ±SD, *P
    Ionomycin, supplied by Abcam, used in various techniques. Bioz Stars score: 95/100, based on 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Tocris ionomycin
    AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro. Naive CD4 + T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + <t>Ionomycin</t> and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P
    Ionomycin, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Cell Signaling Technology Inc ionomycin
    The Lys-63-linked ubiquitylation of inhibitor of differentiation proteins are essential to their autophagic degradation. a <t>Ionomycin</t> and EB1089 enhanced Id-1/2 ubiquitination. HEK293T cells were transiently transfected with the indicated plasmids, treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then incubated in 100 nM Baf A1 for 4 h. Immunoprecipitated Myc-Id-1/2 was subjected to anti-ubiquitin western blotting. b Ionomycin and EB1089 enhanced endogenous Id-1 ubiquitination. SK-N-SH cells were treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then incubated in 100 nM Baf A1 for 4 h. Immunoprecipitated Id-1 was subjected to anti-ubiquitin western blotting. c Id-1 and Id-2 interact with p62. HEK293T cells treated with 6 μM ionomycin or 100 nM EB1089 for 24 h and 100 nM Baf A1 for 6 h before collect. The protein extracts were incubated with anti-p62 or normal mouse IgG antibodies prior to western blot. The resulting immunoprecipitates (IP) were subjected to western blot analysis using the indicated antibodies. WCL, whole-cell lysate. d Id-1 was enclosed in LAMP1-positive autolysosomes. MEFs cells were transiently transfected with Myc-Id-1 and then incubated with 6 μM ionomycin or 100 nM EB1089 for 24 h. Confocal images of transfected cells representing the co-localization of Id-1 with the lysosome marker LAMP1. The merged images and relative insets reveal the co-localization. Scale bar, 10 μm. e Id-2 was enclosed in LAMP1-positive autolysosomes. MEFs cells were transiently transfected with Myc-Id-2, then incubated with 6 μM ionomycin or 100 nM EB1089 for 24 h. Confocal images of transfected cells representing the co-localization of Id-2 with the lysosome marker LAMP1. The merged images and relative insets reveal the co-localization. Scale bar, 10 μm. f , g Id-1 and Id-2 was recruited by p62. MEFs cells were incubated with 6 μM ionomycin for 24 h and Baf A1 for 2 h. Confocal images of cells representing the co-localization of Id-1/2 with p62. The yellow puncta of the merged images reveal the co-localization. Scale bar, 10 μm
    Ionomycin, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 91/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Fisher Scientific ionomycin
    Percentage of sample determined to be electroporated (EP) with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM <t>ionomycin.</t> The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant differences in the percent of EP cells. Regression lines represent the fitted cubic splines.
    Ionomycin, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 93/100, based on 115 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Valiant ionomycin
    BSH effect on intracellular markers of systemic NK cells. Following NK cell enrichment, NK cells were stimulated with <t>PMA/Ionomycin</t> and blocked with Brefeldin A for 4hrs. The differences of day2 or day21 and day-1 are shown. Data are presented as mean±std.dev. N = 9–14. Data are shown as whiskers with 10–90 percentiles. *significantly different (p = 0.049), tested with two sample t test.
    Ionomycin, supplied by Valiant, used in various techniques. Bioz Stars score: 92/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    FUJIFILM ionomycin
    Characterization of aberrant T cells in LTAC mice. (A and B) Frequencies (left) and absolute numbers (right) of TCRβ + cells of CD45 + cells from mLNs (A) or cLP (B) of WT (n = 9–10, filled circle) and LTAC mice (n = 9–11, open circle) determined by flow cytometry analysis. Horizontal bars represent mean. (C) (Left and middle plots) Flow cytometry of CD4 + and CD8α + cell subsets in TCRβ + cells. (Right panels) Frequencies of CD4 + cell subset in TCRβ + cells. The plots are representative of at least three independent experiments. mLNs (upper) or cLP (lower) of WT (n = 7–10, filled circle) and LTAC mice (n = 8–10, open circle), determined by flow cytometry analysis. Horizontal bars represent mean. (D) Genomic DNAs from the sorted T cells in each tissue indicated were subjected to PCR amplification to detect flox (= TAK1 WT) or Δ (= TAK1 KO) allele in the TAK1 genomic locus. The DNA size difference between the flox and Δ bands is shown. Specific DNAs for Cre, loaded in each well, were also shown to prove its existence in the genome. (E) Flow cytometry of intracellular cytokines in TCRβ + CD4 + cells from cLP after culture with PMA + <t>Ionomycin</t> for 5 hours in the presence of GolgiStop. The plots are representative of four independent experiments. (F) Frequencies of each subset of cytokine-producing cells in TCRβ + CD4 + cells in cLP of WT (n = 4, filled circle) and LTAC mice (n = 4, open circle), calculated by flow cytometry analysis from (E). Horizontal bars represent mean. (G) Flow cytometry of gut homing receptors by mLN TCRβ + CD4 + cells. The histograms are representative of at least three independent experiments. (H) Frequencies of α4β7 hi , CCR9 + and CD103 + cells of TCRβ + CD4 + cells in the mLNs of WT (n ≧ 5, filled circle) and LTAC mice (n ≧ 5, open circle), determined by flow cytometry analysis from (H). Horizontal bars represent mean. In (A), (B), (C), (F) and (H), unpaired t tests were performed. Statistical significance was indicated by * P
    Ionomycin, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 93/100, based on 171 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Santa Cruz Biotechnology ionomycin
    Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM <t>ionomycin</t> (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.
    Ionomycin, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 120 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Beyotime ionomycin
    Effect of MFN2 on the immune function of Jurkat cells in response to <t>PMA/ionomycin.</t> Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P
    Ionomycin, supplied by Beyotime, used in various techniques. Bioz Stars score: 92/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    InvivoGen ionomycin
    T cell development and stress-induced apoptosis are normal in A1 −/− mice. ( a , left) Representative FACS plots of developing T cells in the thymus of wild-type and A1 −/− mice. T cells develop in the thymus from the CD4/CD8 double-negative (DN) stage, becoming double positive (DP) and then differentiating into either CD4 or CD8 single-positive cells. The DN population is further subdivided into DN1–4 phases: DN1 (CD44 + CD25 − ), DN2 (CD44 + CD25 + ), DN3 (CD44 − CD25 + ) and DN4 (CD44 − CD25 − ). ( a , right) Quantified total cell numbers for developing T cell populations in wild-type and A1 −/− mice. ( b ) Thymocytes harvested from wild-type and A1 −/− mice were put into culture with the following apoptosis-inducing compounds: ABT-737 (737) 0.5 μ M, dexamethasone (Dexa) 0.5 ng/ml, etoposide (Etopo) 0.5 μ g/ml, <t>ionomycin</t> (Iono), 0.1 μ g/ml, PMA 0.5 ng/ml or DMSO as a control. Apoptosis was assessed by FACS analysis after staining with fluorochrome-conjugated Annexin-V and PI (5 μ g/ml) at 24 h post treatment. Data are representative of six mice for each genotype, with each treatment performed in triplicate
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    92
    LC Laboratories ionomycin
    LMP-induced increases in cytosolic [Ca 2+ ]: kinetics and attenuation by high-level LMP. A , B , and D : changes in cytosolic [Ca 2+ ] were measured in fluo 4-loaded BMDCs (WT or Casp1 −/− as indicated) primed with LPS. Baseline fluo 4 fluorescence was recorded for 5 min before stimulation of the cells with the indicated concentrations of LLME or 6 μM <t>ionomycin.</t> D : parallel wells of BMDCs were treated with or without 100 μM CA074Me during the final 2 h of the 4-h LPS priming incubation. In A and B , cytosolic [Ca 2+ ] was calculated based on calibration after Triton X-100 (Tx)-mediated release of intracellular fluo 4 into the 1.5 mM CaCl 2 -containing medium followed by chelation of Ca 2+ with EGTA; data points represent the means ± SE from 4 experiments. In D , time-dependent changes in fluo 4 fluorescence for WT BMDCs are directly plotted; data represent the average ± range of technical duplicates from a single experiment. C : plot of the differential rates of [Ca 2+ ] increase measured between the 10- and 20-min time points from the time courses in A and B ; ** P
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    Image Search Results


    Miro1-R272Q impairs cytosolic calcium handling and increases sensitivity to calcium stress . ( A ) iPSC-derived neurons were loaded with the cytosolic calcium indicator Fluo-4 AM for live-cell imaging. During imaging, cells were treated with 5 μM ionomycin in order to increase cytosolic calcium levels. Imaging was continued for 10 min with a 2 s interval. Images were obtained with a 40× objective. ( B ) Analysis of mean Fluo-4 AM fluorescence intensity (F1/F0) from images shown in A. Data indicated as mean ± SEM. Significance of first time points after treatment calculated by Holm–Sidak multiple t test; * P

    Journal: Human Molecular Genetics

    Article Title: Impaired mitochondrial–endoplasmic reticulum interaction and mitophagy in Miro1-mutant neurons in Parkinson’s disease

    doi: 10.1093/hmg/ddaa066

    Figure Lengend Snippet: Miro1-R272Q impairs cytosolic calcium handling and increases sensitivity to calcium stress . ( A ) iPSC-derived neurons were loaded with the cytosolic calcium indicator Fluo-4 AM for live-cell imaging. During imaging, cells were treated with 5 μM ionomycin in order to increase cytosolic calcium levels. Imaging was continued for 10 min with a 2 s interval. Images were obtained with a 40× objective. ( B ) Analysis of mean Fluo-4 AM fluorescence intensity (F1/F0) from images shown in A. Data indicated as mean ± SEM. Significance of first time points after treatment calculated by Holm–Sidak multiple t test; * P

    Article Snippet: During imaging, cells were treated with 5 μM ionomycin (Sigma-Aldrich, I0634-1MG).

    Techniques: Derivative Assay, Live Cell Imaging, Imaging, Fluorescence

    Mitochondrial integrity, MPT opening, and apoptosis. A) Mitochondrial calcein loading by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl 2 were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 µM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean ± SD, Ctrl/SNCA-Tri: 3.4/4.9, * p = 0.039). B) MPT-induced mitochondrial calcein loss in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl 2 were assayed 1 hr. after treatment with 4 µM staurosporine under NG conditions. MPT opening results in entry of CoCl 2 into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 µm). Inserts: Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 µm). C) HCI automated fluorescence microscopy analysis of MPT in NPCs treated with 4 µM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean ± SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, * p ≤0.01). D) Kinetic evaluation of MPT opening and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS analysis. NPCs treated and prepared as under B) were loaded with 4 µM stauropsporine and changes in calcein signal normalized to cell number and mitochondrial content (Δ Norm. RFU) were recorded every 1 min for 20 min (n = 8, mean ± SD, Ctrl/SNCA-Tri, HG: −0.06/−0.12, HG+R: −0.17/−0.28, HG+Iono: −0.03/−0.04, * p ≤0.01). E) Cytochrome c immuno-cytochemistry in Ctrl and SNCA-tri NPCs challenged with 200 µM paraquat (PQ) 15 min. before fixation. Shown are permeabilized cells probed with cytochrome c antibody, detected by an Alexa-488 nm labeled secondary antibody (green). Cells were counter stained with Hoechst 33342 (blue) (Scale bar: 100 µm, insert: 10 µm). F) Immunoblot analysis of cytochrome c levels in sub-cellular fractions containing either cellular organelles (containing bound cytochrome c) or cytosolic proteins (with soluble cytochrome c) from NPC cell lysates (Ctrl and SNCA-Tri) treated with paraquat (PQ) as under E). Cytochrome c (14 kDa) and GAPDH (40 kDa) specific antibodies were detected by a secondary IR-dye conjugate.

    Journal: PLoS ONE

    Article Title: Higher Vulnerability and Stress Sensitivity of Neuronal Precursor Cells Carrying an Alpha-Synuclein Gene Triplication

    doi: 10.1371/journal.pone.0112413

    Figure Lengend Snippet: Mitochondrial integrity, MPT opening, and apoptosis. A) Mitochondrial calcein loading by fluorescent plate reader HTS of in NPCs grown in 96 well micro plates. Relative fluorescent signal intensities (RFU) for calcein acquired after 30 min loading with Calcein AM and CoCl 2 were normalized to mitochondrial content (Mitotracker) and to cell number by Hoechst 33342 (H33342). 1 µM ionomycin was added directly before HTS analysis as negative control (Iono) (n = 8, mean ± SD, Ctrl/SNCA-Tri: 3.4/4.9, * p = 0.039). B) MPT-induced mitochondrial calcein loss in Ctrl and SNCA-Tri NPCs after mitochondrial calcein–AM loading. Representative fluorescence microscopy images of Ctrl and SNCA-Tri NPCs loaded with calcein (green), Mitotracker (red) and CoCl 2 were assayed 1 hr. after treatment with 4 µM staurosporine under NG conditions. MPT opening results in entry of CoCl 2 into mitochondria and loss of calcein signal (nuclear counter stain: Hoechst 33342; scale bar: 100 µm). Inserts: Higher magnification images obtained by conventional fluorescence microscopy (Scale bar: 10 µm). C) HCI automated fluorescence microscopy analysis of MPT in NPCs treated with 4 µM staurosporine as under B). Images (see B) were analyzed using MetaXpress image processing software. Depicted are data of cellular calcein signal intensities normalized to mitochondrial content (Norm. RFU Calcein/RFU Mitotracker) from two replicate wells with four image sites/well per treatment condition (n = 16, mean ± SD, Ctrl/SNCA-Tri, HG: 834/457, HG+R: 1425/1011, NG: 864/574, HG+Iono: 187/190, * p ≤0.01). D) Kinetic evaluation of MPT opening and loss of mitochondrial calcein signal after induction of MTP using fluorescence plate reader based HTS analysis. NPCs treated and prepared as under B) were loaded with 4 µM stauropsporine and changes in calcein signal normalized to cell number and mitochondrial content (Δ Norm. RFU) were recorded every 1 min for 20 min (n = 8, mean ± SD, Ctrl/SNCA-Tri, HG: −0.06/−0.12, HG+R: −0.17/−0.28, HG+Iono: −0.03/−0.04, * p ≤0.01). E) Cytochrome c immuno-cytochemistry in Ctrl and SNCA-tri NPCs challenged with 200 µM paraquat (PQ) 15 min. before fixation. Shown are permeabilized cells probed with cytochrome c antibody, detected by an Alexa-488 nm labeled secondary antibody (green). Cells were counter stained with Hoechst 33342 (blue) (Scale bar: 100 µm, insert: 10 µm). F) Immunoblot analysis of cytochrome c levels in sub-cellular fractions containing either cellular organelles (containing bound cytochrome c) or cytosolic proteins (with soluble cytochrome c) from NPC cell lysates (Ctrl and SNCA-Tri) treated with paraquat (PQ) as under E). Cytochrome c (14 kDa) and GAPDH (40 kDa) specific antibodies were detected by a secondary IR-dye conjugate.

    Article Snippet: Cells were treated for 1 hr before measurement with the following ROS generators, apoptosis inducers and mitochondrial inhibitors: 200 µM tert-butyl-hydroxy-peroxide (TBHP) (Sigma, #458139) for 1 hr, 100 µM paraquat (PQ) (Fluka, #36541), 2 µM oligomycin (O) (Sigma, #O4876) for 2 hrs, 1 µM ionomycin (Iono) (Sigma #I0634) or 2 µM CCCP (Sigma, #C2759) for 1 hr.

    Techniques: Negative Control, Fluorescence, Microscopy, Staining, Software, Immunocytochemistry, Labeling

    Increased Tregs frequency in peripheral blood of CLL patients. a Gating strategy used to identify Tregs as CD4 + CD25 high FoxP3 + . Representative box plots of b Tregs frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15); c IL-10-secreting Tregs frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15); d Tregs subsets frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15), all after in vitro priming with IL-6 and phorbol 12-myristate 13-acetate (P), ionomycin (I) and monensin (M). All results are expressed as median and interquartile range. P value shown is obtained from the comparison between the indicated groups by exact non-parametric Mann–Whitney U test (*P

    Journal: Journal of Translational Medicine

    Article Title: Immunosuppressive Treg cells acquire the phenotype of effector-T cells in chronic lymphocytic leukemia patients

    doi: 10.1186/s12967-018-1545-0

    Figure Lengend Snippet: Increased Tregs frequency in peripheral blood of CLL patients. a Gating strategy used to identify Tregs as CD4 + CD25 high FoxP3 + . Representative box plots of b Tregs frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15); c IL-10-secreting Tregs frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15); d Tregs subsets frequency in PBMCs obtained from HV (n = 15) and CLL patients (n = 15), all after in vitro priming with IL-6 and phorbol 12-myristate 13-acetate (P), ionomycin (I) and monensin (M). All results are expressed as median and interquartile range. P value shown is obtained from the comparison between the indicated groups by exact non-parametric Mann–Whitney U test (*P

    Article Snippet: CD4+ cells were primed for 24 h at 37 °C with IL-6 (30 ng/ml, Miltenyi Biotec) overnight (o/n) and then incubated for 5 h at 37 °C with phorbol 12-myristate-13-acetate (P) (50 ng/ml), ionomycin (I) (1 μg/ml, Invitrogen) and GolgiStop Protein Transport Inhibitor (Monensin, BD recommended concentration) (M) based on polarization method previously reported by Musuraca et al. [ ].

    Techniques: In Vitro, MANN-WHITNEY

    Curcumin suppresses CD69 expression in activated T cells. A and B , curcumin suppresses CD69 expression in T cells. Jurkat ( A ) and freshly isolated human peripheral blood T cells ( B ) were stimulated with PMA (5 ng/ml) and ionomycin (1 μ m ) in the

    Journal: The Journal of Biological Chemistry

    Article Title: Curcumin Suppresses T Cell Activation by Blocking Ca2+ Mobilization and Nuclear Factor of Activated T Cells (NFAT) Activation

    doi: 10.1074/jbc.M111.318733

    Figure Lengend Snippet: Curcumin suppresses CD69 expression in activated T cells. A and B , curcumin suppresses CD69 expression in T cells. Jurkat ( A ) and freshly isolated human peripheral blood T cells ( B ) were stimulated with PMA (5 ng/ml) and ionomycin (1 μ m ) in the

    Article Snippet: Jurkat T cells stimulated with PMA/ionomycin for 30 min in the presence or absence of curcumin were fixed with Cytofix/Cytoperm (BD Pharmingen) and washed with Perm/Washing solution (BD Pharmingen) for three times at 4 °C.

    Techniques: Expressing, Isolation

    Curcumin enhances the immunosuppressive activities of CsA. A , curcumin and CsA synergize to inhibit CD69 expression in activated T cells. Jurkat T cells were stimulated with PMA/ionomycin in the absence or the presence of either CsA alone or CsA plus

    Journal: The Journal of Biological Chemistry

    Article Title: Curcumin Suppresses T Cell Activation by Blocking Ca2+ Mobilization and Nuclear Factor of Activated T Cells (NFAT) Activation

    doi: 10.1074/jbc.M111.318733

    Figure Lengend Snippet: Curcumin enhances the immunosuppressive activities of CsA. A , curcumin and CsA synergize to inhibit CD69 expression in activated T cells. Jurkat T cells were stimulated with PMA/ionomycin in the absence or the presence of either CsA alone or CsA plus

    Article Snippet: Jurkat T cells stimulated with PMA/ionomycin for 30 min in the presence or absence of curcumin were fixed with Cytofix/Cytoperm (BD Pharmingen) and washed with Perm/Washing solution (BD Pharmingen) for three times at 4 °C.

    Techniques: Expressing

    Itk signaling is critical for regulating the differentiation and effector function of CD4 + T cells during EAE. A , B , Splenocytes isolated from day 10, day 17, and day 31 immunized WT and Itk −/− mice were stimulated with 5 μg/ml MOG peptide for 72 h. The cells were then restimulated with 10 μg/ml MOG peptide in the presence of Brefeldin A for 5 h, and CD4 + T cells were analyzed for the expression of IFNγ and IL-17A by FACS. Representative flow plots from day 10 is shown in A . B , Cytokine-producing cells quantified for IFNγ (left) or IL-17A (right) stimulation index (fold change in percentage of IFNγ or IL-17A-producing cells in response to MOG peptide compared with media controls). C , Cell culture supernatants of cells treated as in A were analyzed for the levels of IFNγ and IL-17A protein by ELISA. D , Splenocytes from A were stimulated with PMA/ionomycin (50 ng/1 μg/ml; Sigma) in the presence of Brefeldin A for 5 h and quantified for IFNγ (left) or IL-17A (right) stimulation index (fold change in percentage of IFNγ or IL-17A-producing cells in response to MOG peptide compared with media controls). E , Splenocytes obtained from day 17 immunized WT and Itk −/− mice were loaded with CFSE and stimulated with 5 μg/ml MOG peptide for 72 h. Alternatively H 3 -thymidine was added during the last 18 h of culture. CFSE-labeled cells were analyzed for proliferation of CD4 + T cells by FACS (top row), and data are represented as percentage CFSE diluted or fold increase in thymidine incorporation over media control (bottom row). Values are means ± SEMs, * p

    Journal: The Journal of Neuroscience

    Article Title: Itk Signals Promote Neuroinflammation by Regulating CD4+ T-Cell Activation and Trafficking

    doi: 10.1523/JNEUROSCI.1957-14.2015

    Figure Lengend Snippet: Itk signaling is critical for regulating the differentiation and effector function of CD4 + T cells during EAE. A , B , Splenocytes isolated from day 10, day 17, and day 31 immunized WT and Itk −/− mice were stimulated with 5 μg/ml MOG peptide for 72 h. The cells were then restimulated with 10 μg/ml MOG peptide in the presence of Brefeldin A for 5 h, and CD4 + T cells were analyzed for the expression of IFNγ and IL-17A by FACS. Representative flow plots from day 10 is shown in A . B , Cytokine-producing cells quantified for IFNγ (left) or IL-17A (right) stimulation index (fold change in percentage of IFNγ or IL-17A-producing cells in response to MOG peptide compared with media controls). C , Cell culture supernatants of cells treated as in A were analyzed for the levels of IFNγ and IL-17A protein by ELISA. D , Splenocytes from A were stimulated with PMA/ionomycin (50 ng/1 μg/ml; Sigma) in the presence of Brefeldin A for 5 h and quantified for IFNγ (left) or IL-17A (right) stimulation index (fold change in percentage of IFNγ or IL-17A-producing cells in response to MOG peptide compared with media controls). E , Splenocytes obtained from day 17 immunized WT and Itk −/− mice were loaded with CFSE and stimulated with 5 μg/ml MOG peptide for 72 h. Alternatively H 3 -thymidine was added during the last 18 h of culture. CFSE-labeled cells were analyzed for proliferation of CD4 + T cells by FACS (top row), and data are represented as percentage CFSE diluted or fold increase in thymidine incorporation over media control (bottom row). Values are means ± SEMs, * p

    Article Snippet: To stain for intracellular cytokines and transcription factors, cells were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin (50 ng/1 μg/ml; Sigma) and Brefeldin A (Sigma) for 5 h. To examine antigen-specific recall responses, single-cell suspensions of splenocytes or lymph node cells were cultured with the indicated concentration of MOG peptide for 72 h and restimulated with either 10 μg/ml peptide from MOG35–55 or with PMA/ionomycin (50 ng/1 μg/ml; Sigma) in the presence of Brefeldin A (Sigma) for 5 h. Cells were then fixed/permeabilized using the forkhead box p3 (Foxp3) fixation/permeabilization kit and stained with the indicated antibodies (or isotype controls) against surface/intracellular proteins.

    Techniques: Isolation, Mouse Assay, Expressing, FACS, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Labeling

    Th1 and Th17 effector cells are decreased in the CNS of Itk −/− mice. A , Gating strategy to identify cytokine-producing cells and Foxp3 + Treg cells. B , C , Cells isolated from the brain and spinal cord of the indicated day 17 ( B ) and day 31 ( C ) immunized mice were stimulated with PMA/ionomycin in the presence of Brefeldin A for 5 h, and CD4 + T cells were analyzed for the expression of IFNγ and IL-17A by FACS (top rows) and quantified for number of cytokine-producing cells (bottom rows). Values are means ± SEMs, n = 3–5, * p

    Journal: The Journal of Neuroscience

    Article Title: Itk Signals Promote Neuroinflammation by Regulating CD4+ T-Cell Activation and Trafficking

    doi: 10.1523/JNEUROSCI.1957-14.2015

    Figure Lengend Snippet: Th1 and Th17 effector cells are decreased in the CNS of Itk −/− mice. A , Gating strategy to identify cytokine-producing cells and Foxp3 + Treg cells. B , C , Cells isolated from the brain and spinal cord of the indicated day 17 ( B ) and day 31 ( C ) immunized mice were stimulated with PMA/ionomycin in the presence of Brefeldin A for 5 h, and CD4 + T cells were analyzed for the expression of IFNγ and IL-17A by FACS (top rows) and quantified for number of cytokine-producing cells (bottom rows). Values are means ± SEMs, n = 3–5, * p

    Article Snippet: To stain for intracellular cytokines and transcription factors, cells were stimulated with phorbol 12-myristate 13-acetate (PMA)/ionomycin (50 ng/1 μg/ml; Sigma) and Brefeldin A (Sigma) for 5 h. To examine antigen-specific recall responses, single-cell suspensions of splenocytes or lymph node cells were cultured with the indicated concentration of MOG peptide for 72 h and restimulated with either 10 μg/ml peptide from MOG35–55 or with PMA/ionomycin (50 ng/1 μg/ml; Sigma) in the presence of Brefeldin A (Sigma) for 5 h. Cells were then fixed/permeabilized using the forkhead box p3 (Foxp3) fixation/permeabilization kit and stained with the indicated antibodies (or isotype controls) against surface/intracellular proteins.

    Techniques: Mouse Assay, Isolation, Expressing, FACS

    Idelalisib impairs calcium flux and TREM-1 signaling in neutrophils in vitro . PMN from healthy human donors were isolated, preincubated with idelalisib 1 µg/ml and loaded with FLUO-3/AM (both at 37 °C for 30 min) for calcium flux assay. ( A – C ) Fluorescence signals were detected by flow cytometry for 30 s. For PMN activation indicated stimuli (( A ) anti-TREM-1, ( B ) isotype matched control mAb, ( C ) ionomycin) was added. For cell stimulation via TREM-1 receptor (and isotype control) cross-linking was performed with a secondary antibody again after 30 s; fluorescence signals were gained for further 5 min. Ionomycin served as positive control. One out of three experiments is depicted. ( D ) For protein analysis PMN were activated with indicated stimuli (anti-TREM-1 antibody, matched control mAb, LPS) for 30 min after preincubated with idelalisib as described above. Proteins were extracted with an urea-based lysis buffer, SDS PAGE was performed and proteins were blotted by a semi-dry process. Protein activation was analyzed by staining of phosphorylated and non-phosphorylated proteins. ß-actin served as loading control. One out of three experiments is depicted.

    Journal: Scientific Reports

    Article Title: Idelalisib impairs TREM-1 mediated neutrophil inflammatory responses

    doi: 10.1038/s41598-018-23808-2

    Figure Lengend Snippet: Idelalisib impairs calcium flux and TREM-1 signaling in neutrophils in vitro . PMN from healthy human donors were isolated, preincubated with idelalisib 1 µg/ml and loaded with FLUO-3/AM (both at 37 °C for 30 min) for calcium flux assay. ( A – C ) Fluorescence signals were detected by flow cytometry for 30 s. For PMN activation indicated stimuli (( A ) anti-TREM-1, ( B ) isotype matched control mAb, ( C ) ionomycin) was added. For cell stimulation via TREM-1 receptor (and isotype control) cross-linking was performed with a secondary antibody again after 30 s; fluorescence signals were gained for further 5 min. Ionomycin served as positive control. One out of three experiments is depicted. ( D ) For protein analysis PMN were activated with indicated stimuli (anti-TREM-1 antibody, matched control mAb, LPS) for 30 min after preincubated with idelalisib as described above. Proteins were extracted with an urea-based lysis buffer, SDS PAGE was performed and proteins were blotted by a semi-dry process. Protein activation was analyzed by staining of phosphorylated and non-phosphorylated proteins. ß-actin served as loading control. One out of three experiments is depicted.

    Article Snippet: Ionomycin calcium salt from Streptomyces conglobatus (1 µM; Sigma-Aldrich, Taufkirchen, Germany) served as positive control.

    Techniques: In Vitro, Isolation, Calcium Flux Assay, Fluorescence, Flow Cytometry, Cytometry, Activation Assay, Cell Stimulation, Positive Control, Lysis, SDS Page, Staining

    NKT1 cells negatively correlate with arthritis severity in SKG mice. a Representative flow cytometry plot of cytokine profile of iNKT (TCRβ int CD1d-αGC tetramer + ) cells from joint-draining lymph nodes and joints (wrists and ankles) of arthritic SKG mice stimulated with PMA and ionomycin. b Representative flow cytometry of iNKT (TCRβ int CD1d-αGC tetramer + ) cell subsets in ankles during the course of mannan-induced arthritis in male SKG mice. Day 0 (circle), day 12 (square), day 22 (triangle), and day 47 (diamond) post-mannan injection. c Frequency of NKT1 (T-bet + RORγt − , left, green) and NKT17 cells (T-bet − RORγt + , right, purple) during the course of mannan-induced arthritis in SKG mice. d Correlation between NKT1 (left panel, green) and NKT17 (right panel, purple) and changes in ankle swelling in mice with mannan-induced arthritis. e Representative flow cytometry plot of iNKT cells (CD3 + Vα24Jα18 + (6B11)) in paired peripheral blood (PB) and synovial fluid (SF) samples derived from rheumatoid arthritis (RA) patients ( n = 8). f Levels of IFNγ in paired peripheral blood (PB) and synovial fluid (SF) samples stimulated with αGC (RA patients, n = 4). g Representative histogram (of a total of 5 RA patients) of IFN-γ expressing iNKT cells (CD3 + Vα24Jα18 + (6B11 antibody-reactive) after stimulation with PMA and ionomycin (black/red histogram) or unstimulated control (filled histogram). h Expression of IFN-γ and IL-4 from splenic iNKT cells after injection of αGC in WT BALB/c (blue) or SKG mice (red). i Expression of IFN-γ by splenic iNKT cells after injection of LPS in WT BALB/c (blue) or SKG mice (red). j Serum IFNγ in WT BALB/c (blue) or SKG mice (red) after LPS injection. k Production of IFN-γ by iNKT cells after stimulation of lymph node cultures for 48 h with IL-12 (20 ng/ml). Lymph node cultures prepared from WT BALB/c (blue), pre-arthritic SKG (red, 7–8 weeks old) and arthritic SKG mice (black, 40 days post-mannan injection). Representative of at least two independent experiments are shown. Graphs represent mean ± SEM with symbols representing individual mice. * p

    Journal: Nature Communications

    Article Title: Altered thymic differentiation and modulation of arthritis by invariant NKT cells expressing mutant ZAP70

    doi: 10.1038/s41467-018-05095-7

    Figure Lengend Snippet: NKT1 cells negatively correlate with arthritis severity in SKG mice. a Representative flow cytometry plot of cytokine profile of iNKT (TCRβ int CD1d-αGC tetramer + ) cells from joint-draining lymph nodes and joints (wrists and ankles) of arthritic SKG mice stimulated with PMA and ionomycin. b Representative flow cytometry of iNKT (TCRβ int CD1d-αGC tetramer + ) cell subsets in ankles during the course of mannan-induced arthritis in male SKG mice. Day 0 (circle), day 12 (square), day 22 (triangle), and day 47 (diamond) post-mannan injection. c Frequency of NKT1 (T-bet + RORγt − , left, green) and NKT17 cells (T-bet − RORγt + , right, purple) during the course of mannan-induced arthritis in SKG mice. d Correlation between NKT1 (left panel, green) and NKT17 (right panel, purple) and changes in ankle swelling in mice with mannan-induced arthritis. e Representative flow cytometry plot of iNKT cells (CD3 + Vα24Jα18 + (6B11)) in paired peripheral blood (PB) and synovial fluid (SF) samples derived from rheumatoid arthritis (RA) patients ( n = 8). f Levels of IFNγ in paired peripheral blood (PB) and synovial fluid (SF) samples stimulated with αGC (RA patients, n = 4). g Representative histogram (of a total of 5 RA patients) of IFN-γ expressing iNKT cells (CD3 + Vα24Jα18 + (6B11 antibody-reactive) after stimulation with PMA and ionomycin (black/red histogram) or unstimulated control (filled histogram). h Expression of IFN-γ and IL-4 from splenic iNKT cells after injection of αGC in WT BALB/c (blue) or SKG mice (red). i Expression of IFN-γ by splenic iNKT cells after injection of LPS in WT BALB/c (blue) or SKG mice (red). j Serum IFNγ in WT BALB/c (blue) or SKG mice (red) after LPS injection. k Production of IFN-γ by iNKT cells after stimulation of lymph node cultures for 48 h with IL-12 (20 ng/ml). Lymph node cultures prepared from WT BALB/c (blue), pre-arthritic SKG (red, 7–8 weeks old) and arthritic SKG mice (black, 40 days post-mannan injection). Representative of at least two independent experiments are shown. Graphs represent mean ± SEM with symbols representing individual mice. * p

    Article Snippet: For evaluation of IFN-γ production by iNKT cells directly ex vivo, freshly isolated PBMC and SFMC were incubated for 4 h with phorbol 12-myristate 13-acetate (PMA, 25 ng/ml), calcium-ionomycin (CaI, 1 µg/ml; both from Sigma) and brefeldin A (BFA, 10 ng/ml; BD) or BFA alone (negative control).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Injection, Derivative Assay, Expressing

    Itch and WWP2 collaboratively regulate T H 2 differentiation. a , Frequency of splenic memory-like (CD44 hi CD62L − ) CD4 + T cells (left) and naive (CD25 − CD44 − CD62L hi ) CD4 + T cells (middle) from 6- to 7-week-old wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre and DKO mice (key; n = 8 per group), and flow cytometry of splenic naive CD4 + T cells from such mice, showing gating used (right). Numbers adjacent to outlined areas (right) indicate percent CD4 + CD25 − CD44 − CD62L hi cells. b , Gene-expression profiles (left) of splenic CD4 + T cells stimulated for 4h with PMA and ionomycin, showing genes expressed differentially (mean of normalized counts) in Wwp2 −/− , Itch f/f Cd4 -Cre or DKO cells (above plots) relative to their expression in wild-type cells, and expression of genes encoding selected cytokines (middle) or transcriptional regulators (right) in cells as at left (above plots), presented as log 2 reads per kilobase of exon per million mapped reads (RPKM) values. c , Frequency of CD4 + T cells with intracellular staining of IL-4 (left) or IFN-γ (right) among splenocytes obtained from mice as in a (key; n = 5–6 per group (IL-4) or n = 4–5 per group (IFN-γ)) and stimulated for 5h in vitro with PMA and ionomycin in the presence of the protein-transport inhibitor GolgiStop. d , ELISA of IL-4 (left) or IFN-γ (right) in supernatants of CD4 + T cells sorted from mice as in a (key; n = 3 per group) and stimulated for 48h in vitro with anti-CD3 plus anti-CD28. e , f , Immunoblot analysis of GATA-3 ( e ) and phosphorylated (p-) and total STAT6 ( e ) or STAT5 ( f ), as well as actin (loading control) (left margin), in lysates of CD4 + T cells sorted from mice as in a (above lanes) and left unstimulated (left) or stimulated for 16h with anti-CD3 plus anti-CD28 (right; α-CD3 + α-CD28); numbers below lanes indicate the ratio of phosphorylated protein to total protein. Each symbol ( a , c , d ) represents an individual mouse; small horizontal lines ( a ) indicate the mean (±s.d.). * P

    Journal: Nature immunology

    Article Title: The E3 ligases Itch and WWP2 cooperate to limit TH2 differentiation by enhancing signaling through the TCR

    doi: 10.1038/s41590-018-0137-8

    Figure Lengend Snippet: Itch and WWP2 collaboratively regulate T H 2 differentiation. a , Frequency of splenic memory-like (CD44 hi CD62L − ) CD4 + T cells (left) and naive (CD25 − CD44 − CD62L hi ) CD4 + T cells (middle) from 6- to 7-week-old wild-type, Wwp2 −/− , Itch f/f Cd4 -Cre and DKO mice (key; n = 8 per group), and flow cytometry of splenic naive CD4 + T cells from such mice, showing gating used (right). Numbers adjacent to outlined areas (right) indicate percent CD4 + CD25 − CD44 − CD62L hi cells. b , Gene-expression profiles (left) of splenic CD4 + T cells stimulated for 4h with PMA and ionomycin, showing genes expressed differentially (mean of normalized counts) in Wwp2 −/− , Itch f/f Cd4 -Cre or DKO cells (above plots) relative to their expression in wild-type cells, and expression of genes encoding selected cytokines (middle) or transcriptional regulators (right) in cells as at left (above plots), presented as log 2 reads per kilobase of exon per million mapped reads (RPKM) values. c , Frequency of CD4 + T cells with intracellular staining of IL-4 (left) or IFN-γ (right) among splenocytes obtained from mice as in a (key; n = 5–6 per group (IL-4) or n = 4–5 per group (IFN-γ)) and stimulated for 5h in vitro with PMA and ionomycin in the presence of the protein-transport inhibitor GolgiStop. d , ELISA of IL-4 (left) or IFN-γ (right) in supernatants of CD4 + T cells sorted from mice as in a (key; n = 3 per group) and stimulated for 48h in vitro with anti-CD3 plus anti-CD28. e , f , Immunoblot analysis of GATA-3 ( e ) and phosphorylated (p-) and total STAT6 ( e ) or STAT5 ( f ), as well as actin (loading control) (left margin), in lysates of CD4 + T cells sorted from mice as in a (above lanes) and left unstimulated (left) or stimulated for 16h with anti-CD3 plus anti-CD28 (right; α-CD3 + α-CD28); numbers below lanes indicate the ratio of phosphorylated protein to total protein. Each symbol ( a , c , d ) represents an individual mouse; small horizontal lines ( a ) indicate the mean (±s.d.). * P

    Article Snippet: For intracellular cytokine staining, cells were stimulated with PMA and ionomycin in the presence of BD GoligiStop (BD Bioscience).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Expressing, Staining, In Vitro, Enzyme-linked Immunosorbent Assay

    Effect of PKC and Ca 2+ and c-Src on Pyk2 Activation Acini were stimulated for 5 min with Cch (10 −4 M), PMA (10 −6 M), Cch and 2 mM EGTA, and ionomycin (10 −7 M). The amount of phosphorylated Pyk2 (Tyr 402 )/total Pyk2 is shown in A . Data are mean ± SEM from 3–6 independent experiments. Acini were also preincubated with PP1 for 15 min prior to stimulation with Cch. The amount of phosphorylated Pyk (Tyr 402 ) is shown in B . Data are mean ± SEM from 4 independent experiments. * indicates statistical significance from basal. # denotes statistical significance from Cch alone.

    Journal: FEBS letters

    Article Title: Role of Protein kinase C, Ca2+, Pyk2 and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK.

    doi:

    Figure Lengend Snippet: Effect of PKC and Ca 2+ and c-Src on Pyk2 Activation Acini were stimulated for 5 min with Cch (10 −4 M), PMA (10 −6 M), Cch and 2 mM EGTA, and ionomycin (10 −7 M). The amount of phosphorylated Pyk2 (Tyr 402 )/total Pyk2 is shown in A . Data are mean ± SEM from 3–6 independent experiments. Acini were also preincubated with PP1 for 15 min prior to stimulation with Cch. The amount of phosphorylated Pyk (Tyr 402 ) is shown in B . Data are mean ± SEM from 4 independent experiments. * indicates statistical significance from basal. # denotes statistical significance from Cch alone.

    Article Snippet: Ionomycin was purchased from Alexis Biochemicals (San Diego, CA).

    Techniques: Activation Assay

    Effect of Ionomycin on p42/p44 MAPK Activation Acini were incubated for 10 min with increasing concentrations of ionomycin (10 −9 – 10 −5 M). Activated p42/p44 MAPK/total p42 MAPK was analyzed via western blot. Representative experiment is shown in ( A ). The results of 5 independent experiments are shown in ( B ). Data are mean ± SEM. * indicates statistical significance from basal.

    Journal: FEBS letters

    Article Title: Role of Protein kinase C, Ca2+, Pyk2 and c-Src in Agonist Activation of Rat Lacrimal Gland p42/p44 MAPK.

    doi:

    Figure Lengend Snippet: Effect of Ionomycin on p42/p44 MAPK Activation Acini were incubated for 10 min with increasing concentrations of ionomycin (10 −9 – 10 −5 M). Activated p42/p44 MAPK/total p42 MAPK was analyzed via western blot. Representative experiment is shown in ( A ). The results of 5 independent experiments are shown in ( B ). Data are mean ± SEM. * indicates statistical significance from basal.

    Article Snippet: Ionomycin was purchased from Alexis Biochemicals (San Diego, CA).

    Techniques: Activation Assay, Incubation, Western Blot

    TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or Ionomycin ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).

    Journal: bioRxiv

    Article Title: A Novel PHOX/CD38/MCOLN1/TFEB Axis Important For Macrophage Activation During Bacterial Phagocytosis

    doi: 10.1101/669325

    Figure Lengend Snippet: TFEB activation is mediated by TRPML1/MCOLN1, Ca 2+ , and calcineurin. A-E. GFP-TFEB iBMDMs were treated with DMSO without infection (t = 3 h, A ) or treated for 3 h and subsequently infected with S. aureus ( B , MOI = 10, t = 3 h). In parallel, cells were treated with BAPTA ( C, 10 μM, t = 3 h) or FK506 ( D, 5 μM, t = 6 h) and subsequently infected with S. aureus (MOI = 10, t = 3 h). E. Quantification of GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s posthoc test). F-K. GFP-TFEB iBMDMs were treated with scrambled (Scr, F ) or siRNA against Ppp3cb ( G ), Ppp3r1 ( H ) for 48 h. Cells were treated with Scr ( I ), Ppp3cb ( J ), or Ppp3r1 ( K ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). L. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 210 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). M, N. GFP-TFEB iBMDMs were treated with DMSO ( M , t = 6 h) or Ionomycin ( N , 10 μM, t = 6 h). O. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 350 cells). **** p ≤ 0.0001 (one-way ANOVA followed by Tukey’s post-hoc test). P-S. GFP-TFEB iBMDMs were treated with scrambled (Scr, P ) or siRNA against Mcoln1 ( Q ) for 48 h. Cells were treated with Scr ( R ) or Mcoln1 ( S ) siRNA for 48 h prior to infection with S. aureus (MOI = 10, t = 3 h). T. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 300 cells). *** p ≤ 0.001 (one-way ANOVA followed by Tukey’s post-hoc test). U, V. GFP-TFEB iBMDMs were treated with DMSO ( U , t = 3 h) or ML-SA1 ( V , 10 μM, t = 3 h). W. GFP-TFEB N/C Ratio by CellProfiler (3 biological replicates, n = 355 cells). **** p ≤ 0.0001 (two-sample two-sided t test).

    Article Snippet: Drugs and reagents Lipopolysaccharides (LPS) from S. enterica serotype Typhimurium (Sigma-Aldrich, L6143-1MG, 10ug/ml): Peptidoglycan (PGN) from S. aureus (Invivogen, tlrl-pgns2): Monophosphoryl Lipid A (MPLA-SM) (Invivogen, tlrl-mpla): ML-SA1 (Sigma Aldrich, SML0627) Ionomycin (Cayman Chemical Company, 10004974): FK-506 (Cayman Chemical Company, 10007965): BAPTA AM (Cayman Chemical Company, 15551): Ned-19 (Cayman Chemical Company, 17527): Kuromanin (Cayman Chemical Company, 16406): Apigenin (Cayman Chemical Company, 10010275): CCCP (Cayman Chemical Company, 25458): N-acetyl-L-Cysteine (NAC) (Cayman Chemical Company, 20261): N-acetyl-L-Cysteine amide (NACA) (Cayman Chemical Company, 25866): Apocynin (Cayman Chemical Company, 11976): D609 (Cayman Chemical, 13307, 50 µM): kb NB 142-70 (Cayman Chemical Company, 18002).

    Techniques: Activation Assay, Infection

    Characterization of the lymphoid compartment in PbA-infected B6, BXH2, [BXH2×B6]F1 and Irf1 −/− mice. Characterization of the lymphoid compartment was carried out as described in the legend of Figure 6 . Splenocytes were stained with CD45, TCRβ, CD4 and CD8 antibodies and representative cellular profiles are shown for each strain (A), where numbers indicate mean ± SD (gated as percentages of CD45 + cells). Absolute numbers are indicated in dot plots for total spleen CD4 + cells (B) and CD8 + cells (C). (D) Serum IFNγ levels were significantly lower in ECM-resistant BXH2, [BXH2×B6]F1 and Irf1 −/− mice. (E) IFNγ production was assayed in vitro in culture supernatants from infected mice with or without stimulation with PMA/Ionomycin or with IL12p70. The p-values were calculated relative to B6 controls with Student's t-test (**p

    Journal: PLoS Pathogens

    Article Title: Irf8-Regulated Genomic Responses Drive Pathological Inflammation during Cerebral Malaria

    doi: 10.1371/journal.ppat.1003491

    Figure Lengend Snippet: Characterization of the lymphoid compartment in PbA-infected B6, BXH2, [BXH2×B6]F1 and Irf1 −/− mice. Characterization of the lymphoid compartment was carried out as described in the legend of Figure 6 . Splenocytes were stained with CD45, TCRβ, CD4 and CD8 antibodies and representative cellular profiles are shown for each strain (A), where numbers indicate mean ± SD (gated as percentages of CD45 + cells). Absolute numbers are indicated in dot plots for total spleen CD4 + cells (B) and CD8 + cells (C). (D) Serum IFNγ levels were significantly lower in ECM-resistant BXH2, [BXH2×B6]F1 and Irf1 −/− mice. (E) IFNγ production was assayed in vitro in culture supernatants from infected mice with or without stimulation with PMA/Ionomycin or with IL12p70. The p-values were calculated relative to B6 controls with Student's t-test (**p

    Article Snippet: 4×106 cells were plated in 48-well tissue culture plates and were stimulated with PMA/Ionomycin (eBioscience) or IL12p70 (Biolegend) for 48 hours.

    Techniques: Infection, Mouse Assay, Staining, In Vitro

    TH2849 potentially induces autophagy by forming a complex with FKBP12 without interfering to the calcineurin/NFAT and IL2/p34cdc2/cyclin A signal pathway. (A) PC12 cells were pretreated with siRNA against FKBP12 for 48 h, and then, the protein and mRNA levels were examined. Representative images (B) and quantification (C) of PC12 cells with EGFP‐LC3 vesicles (autophagosomes) in the presence or absence of siRNA against FKBP12. PC12 cells co‐transfected with siRNA against FKBP12 and EGFP‐LC3 were treated with 1‰ DMSO, 1 μmol/L rapamycin, 10 μmol/L FK506, 1 μmol/L TH 2451 and 1 μmol/L TH 2849 for 24 h. (D) Representative images of distribution of EGFP‐NFATc1. The EGFP‐NFATc1 transfected MCF‐7 cells were treated with 2 μmol/L ionomycin or co‐treated with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263 and TH2287 for 2 h as indicated. (E ) Quantification of nucleus translocated EGFP‐NFATc1 cells in (D). (F) Representative Western blotting image of p34 cdc2 and cyclin A levels. Factor‐deprived CTLL‐2 cells were cultured for 14 h in basic medium only, 50 units/mL IL‐2 with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263, and TH2287 were added respectively during the last 1 hour. Quantification of relative p34 cdc2 (G) and cyclin A (H) expression levels in (F). All the substances were dissolved in DMSO. Scale bars: 10 μm. Data are shown as the mean ± SEM * P

    Journal: CNS Neuroscience & Therapeutics

    Article Title: Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect, et al. Rapamycin and FK506 derivative TH2849 could ameliorate neurodegenerative diseases through autophagy with low immunosuppressive effect

    doi: 10.1111/cns.13062

    Figure Lengend Snippet: TH2849 potentially induces autophagy by forming a complex with FKBP12 without interfering to the calcineurin/NFAT and IL2/p34cdc2/cyclin A signal pathway. (A) PC12 cells were pretreated with siRNA against FKBP12 for 48 h, and then, the protein and mRNA levels were examined. Representative images (B) and quantification (C) of PC12 cells with EGFP‐LC3 vesicles (autophagosomes) in the presence or absence of siRNA against FKBP12. PC12 cells co‐transfected with siRNA against FKBP12 and EGFP‐LC3 were treated with 1‰ DMSO, 1 μmol/L rapamycin, 10 μmol/L FK506, 1 μmol/L TH 2451 and 1 μmol/L TH 2849 for 24 h. (D) Representative images of distribution of EGFP‐NFATc1. The EGFP‐NFATc1 transfected MCF‐7 cells were treated with 2 μmol/L ionomycin or co‐treated with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263 and TH2287 for 2 h as indicated. (E ) Quantification of nucleus translocated EGFP‐NFATc1 cells in (D). (F) Representative Western blotting image of p34 cdc2 and cyclin A levels. Factor‐deprived CTLL‐2 cells were cultured for 14 h in basic medium only, 50 units/mL IL‐2 with 1‰ DMSO, 1 μmol/L rapamycin, 1 μmol/L FK506, TH2451, TH 2849, TH3263, and TH2287 were added respectively during the last 1 hour. Quantification of relative p34 cdc2 (G) and cyclin A (H) expression levels in (F). All the substances were dissolved in DMSO. Scale bars: 10 μm. Data are shown as the mean ± SEM * P

    Article Snippet: Ionomycin was purchased from Merck.

    Techniques: Transfection, Western Blot, Cell Culture, Expressing

    Action of ionomycin on a csp mutant preparation. In the top ( 21°C ), mejps ( first two traces ) and the ejp are shown in a csp mutant preparation at permissive temperature. The characteristic failure of the ejp, but not the mejps, is illustrated in

    Journal: The Journal of Neuroscience

    Article Title: Evidence That Cysteine String Proteins Regulate an Early Step in the Ca2+-Dependent Secretion of Neurotransmitter atDrosophila Neuromuscular Junctions

    doi: 10.1523/JNEUROSCI.17-19-07203.1997

    Figure Lengend Snippet: Action of ionomycin on a csp mutant preparation. In the top ( 21°C ), mejps ( first two traces ) and the ejp are shown in a csp mutant preparation at permissive temperature. The characteristic failure of the ejp, but not the mejps, is illustrated in

    Article Snippet: For experiments using the calcium ionophore ionomycin (Calbiochem, La Jolla, CA), a stock of ionomycin was prepared in dimethylsulfoxide (Sigma, St. Louis, MO) and diluted to the working concentration (10 μ m ionomycin) in recording solution under conditions in which the final concentration of solvent was < 0.1%.

    Techniques: Mutagenesis

    Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore ionomycin was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.

    Journal: PLoS ONE

    Article Title: Cytotoxicity of Tumor Antigen Specific Human T Cells Is Unimpaired by Arginine Depletion

    doi: 10.1371/journal.pone.0063521

    Figure Lengend Snippet: Arginine-independent T cell functions: chemotaxis and calcium flux. A Purified primary human T cells were preincubated either for 24 h or 48 h in the presence (+Arg, 1000 µM) or absence (−Arg, 0 µM) of arginine. They were then placed in the respective media into the upper compartment of a trans-well plate, whereas a chemokine (SDF-1α) was added to the lower compartment of the well. Wells without chemokine were also set up to detect spontaneous migration of cells. Maximum migration was determined by adding the same number of T cells to the lower compartment of a separate well. After 4 h cells in the lower compartment were quantified flow-cytometrically. Migration of T cells (%) was calculated relatively to the maximum migration which was set to 100%. Shown are summarized mean values and standard deviations of results from 3 individual experiments. NS: not significant. B Calcium signaling in primary human T cells in the presence (+ Arg, 1000 µM) or absence (−Arg) of arginine was analyzed by flow cytometry using the calcium indicating dye Indo 1-AM. The ratio of Indo-1 violet/Indo-1 blue correlates directly with the amount of calcium in the cell. T cells were preincubated with OKT-3 antibody and calcium levels of resting cells were monitored for 3 min. The cells were then stimulated by adding a crosslinking antibody ( = stimulus), which led to increasing calcium levels irrespective of arginine. As a negative control, isotype control antibody was used instead of OKT-3. Finally, the calcium ionophore ionomycin was added as a positive control. One representative analysis (total number of experiments = 3) is shown as a histogram plot overlay.

    Article Snippet: Flow cytometric analysis (LSRII flow cytometer, BD Biosciences) was done as follows: after measurement of unstimulated cells for 3 min, cross-linking goat-anti-mouse-IgG+IgM antibody (final concentration 10 µg/ml, Dianova, Hamburg, Germany) was added and cells were analyzed for additional 3 min. As a positive control, cells were then activated by ionomycin (final concentration 1 µM; Merck, Darmstadt, Germany).

    Techniques: Chemotaxis Assay, Purification, Migration, Flow Cytometry, Cytometry, Negative Control, Positive Control

    Ca 2+ -evoked nNOS- and eNOS-derived NO generation profiles in ionomycin permeabilized cells (A) Mean curves represent NO generation profiles derived from eNOS (blue curve) and nNOS (green curve) in response to Ca 2+ addition. Experiments were performed in the presence of 5 μM ionomycin. NO recordings were performed using C-geNOp. (B) Bars represent respective statistics of panel A, maximum initial slope of nNOS- (green bar, n = 34/3) and eNOS-derived (blue bar, n = 36/3) NO signals. Mean values are shown ±SD, *P

    Journal: Nitric oxide : biology and chemistry

    Article Title: Real-time visualization of distinct nitric oxide generation of nitric oxide synthase isoforms in single cells

    doi: 10.1016/j.niox.2017.09.001

    Figure Lengend Snippet: Ca 2+ -evoked nNOS- and eNOS-derived NO generation profiles in ionomycin permeabilized cells (A) Mean curves represent NO generation profiles derived from eNOS (blue curve) and nNOS (green curve) in response to Ca 2+ addition. Experiments were performed in the presence of 5 μM ionomycin. NO recordings were performed using C-geNOp. (B) Bars represent respective statistics of panel A, maximum initial slope of nNOS- (green bar, n = 34/3) and eNOS-derived (blue bar, n = 36/3) NO signals. Mean values are shown ±SD, *P

    Article Snippet: Ionomycin was obtained from Abcam (Cambridge, UK).

    Techniques: Derivative Assay

    AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro. Naive CD4 + T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + Ionomycin and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P

    Journal: PLoS ONE

    Article Title: mTOR Inhibition Attenuates Dextran Sulfate Sodium-Induced Colitis by Suppressing T Cell Proliferation and Balancing TH1/TH17/Treg Profile

    doi: 10.1371/journal.pone.0154564

    Figure Lengend Snippet: AZD8055 inhibits the proliferation and differentiation of naive CD4+ T cells in vitro. Naive CD4 + T cells were isolated from spleen and lymph nodes of mice, CFSE-labeled, and activated in the presence of AZD8055 (0nM, 20nM, 50nM) under TH0, TH1 and TH17 conditions for 4 days. (A) TH1 cells were activated with PMA + Ionomycin and stained for CD4 and intracellular expression of IFN-γ in the presence of AZD8055. Dot-plots showed the expression of CD4+ IFN-γ+ TH1 cells under TH0 condition (Upper panels). Dot-plots showed the differentiation of TH1 cells under TH1 condition (Middle panels). Scatterplot displayed the proliferation of TH1 cells under TH1 condition (Lower panels). (B) TH17 cells were stimulated with PMA + Ionomycin and stained for CD4 and intracellular expression of IL-17 in the presence of AZD8055. Dot-plots showed the expression of CD4+ IL-17 + TH17 cells under TH0 condition (Upper panels). Scatterplot displayed the differentiation of TH17 cells under TH17 condition (Lower panels). (C) Results were from three independent experiments. All results showed the mean±SEM. Statistical significance was determined by student’s t-test. *P

    Article Snippet: To determine the percentage of TH1 and TH17 cells, mononuclear cells were stimulated with PMA (50ng/mL, sigma) and Ionomycin (1μg/mL, Tocris) and BFA (1:1000, eBioscience) for 6 hours.

    Techniques: In Vitro, Isolation, Mouse Assay, Labeling, Staining, Expressing

    The Lys-63-linked ubiquitylation of inhibitor of differentiation proteins are essential to their autophagic degradation. a Ionomycin and EB1089 enhanced Id-1/2 ubiquitination. HEK293T cells were transiently transfected with the indicated plasmids, treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then incubated in 100 nM Baf A1 for 4 h. Immunoprecipitated Myc-Id-1/2 was subjected to anti-ubiquitin western blotting. b Ionomycin and EB1089 enhanced endogenous Id-1 ubiquitination. SK-N-SH cells were treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then incubated in 100 nM Baf A1 for 4 h. Immunoprecipitated Id-1 was subjected to anti-ubiquitin western blotting. c Id-1 and Id-2 interact with p62. HEK293T cells treated with 6 μM ionomycin or 100 nM EB1089 for 24 h and 100 nM Baf A1 for 6 h before collect. The protein extracts were incubated with anti-p62 or normal mouse IgG antibodies prior to western blot. The resulting immunoprecipitates (IP) were subjected to western blot analysis using the indicated antibodies. WCL, whole-cell lysate. d Id-1 was enclosed in LAMP1-positive autolysosomes. MEFs cells were transiently transfected with Myc-Id-1 and then incubated with 6 μM ionomycin or 100 nM EB1089 for 24 h. Confocal images of transfected cells representing the co-localization of Id-1 with the lysosome marker LAMP1. The merged images and relative insets reveal the co-localization. Scale bar, 10 μm. e Id-2 was enclosed in LAMP1-positive autolysosomes. MEFs cells were transiently transfected with Myc-Id-2, then incubated with 6 μM ionomycin or 100 nM EB1089 for 24 h. Confocal images of transfected cells representing the co-localization of Id-2 with the lysosome marker LAMP1. The merged images and relative insets reveal the co-localization. Scale bar, 10 μm. f , g Id-1 and Id-2 was recruited by p62. MEFs cells were incubated with 6 μM ionomycin for 24 h and Baf A1 for 2 h. Confocal images of cells representing the co-localization of Id-1/2 with p62. The yellow puncta of the merged images reveal the co-localization. Scale bar, 10 μm

    Journal: Nature Communications

    Article Title: CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

    doi: 10.1038/s41467-017-01272-2

    Figure Lengend Snippet: The Lys-63-linked ubiquitylation of inhibitor of differentiation proteins are essential to their autophagic degradation. a Ionomycin and EB1089 enhanced Id-1/2 ubiquitination. HEK293T cells were transiently transfected with the indicated plasmids, treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then incubated in 100 nM Baf A1 for 4 h. Immunoprecipitated Myc-Id-1/2 was subjected to anti-ubiquitin western blotting. b Ionomycin and EB1089 enhanced endogenous Id-1 ubiquitination. SK-N-SH cells were treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then incubated in 100 nM Baf A1 for 4 h. Immunoprecipitated Id-1 was subjected to anti-ubiquitin western blotting. c Id-1 and Id-2 interact with p62. HEK293T cells treated with 6 μM ionomycin or 100 nM EB1089 for 24 h and 100 nM Baf A1 for 6 h before collect. The protein extracts were incubated with anti-p62 or normal mouse IgG antibodies prior to western blot. The resulting immunoprecipitates (IP) were subjected to western blot analysis using the indicated antibodies. WCL, whole-cell lysate. d Id-1 was enclosed in LAMP1-positive autolysosomes. MEFs cells were transiently transfected with Myc-Id-1 and then incubated with 6 μM ionomycin or 100 nM EB1089 for 24 h. Confocal images of transfected cells representing the co-localization of Id-1 with the lysosome marker LAMP1. The merged images and relative insets reveal the co-localization. Scale bar, 10 μm. e Id-2 was enclosed in LAMP1-positive autolysosomes. MEFs cells were transiently transfected with Myc-Id-2, then incubated with 6 μM ionomycin or 100 nM EB1089 for 24 h. Confocal images of transfected cells representing the co-localization of Id-2 with the lysosome marker LAMP1. The merged images and relative insets reveal the co-localization. Scale bar, 10 μm. f , g Id-1 and Id-2 was recruited by p62. MEFs cells were incubated with 6 μM ionomycin for 24 h and Baf A1 for 2 h. Confocal images of cells representing the co-localization of Id-1/2 with p62. The yellow puncta of the merged images reveal the co-localization. Scale bar, 10 μm

    Article Snippet: Ionomycin was purchased from Cell Signaling Technology.

    Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Marker

    The CaMKII-mediated phosphorylation of Beclin 1 at Ser90 induces the differentiation of neuroblastoma cells. a Ionomycin and EB1089 regulate neuroblastoma cell differentiation. Both cell lines were treated with 1.5 μM ionomycin or 50 nM EB1089 for 7 days and then observed using fluorescence microscopy. Values were shown as the means ± SD of 5 random areas. ** P

    Journal: Nature Communications

    Article Title: CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

    doi: 10.1038/s41467-017-01272-2

    Figure Lengend Snippet: The CaMKII-mediated phosphorylation of Beclin 1 at Ser90 induces the differentiation of neuroblastoma cells. a Ionomycin and EB1089 regulate neuroblastoma cell differentiation. Both cell lines were treated with 1.5 μM ionomycin or 50 nM EB1089 for 7 days and then observed using fluorescence microscopy. Values were shown as the means ± SD of 5 random areas. ** P

    Article Snippet: Ionomycin was purchased from Cell Signaling Technology.

    Techniques: Cell Differentiation, Fluorescence, Microscopy

    TRAF-6 induced the K63-linked ubiquitination of Id-1 at the K88 residue. a CaMKII and phosphorylation of Beclin 1 Ser90 are required for K63 ubiquitination of Ids. HEK293T cells were transiently co-transfected with the indicated plasmids, treated with 6 μM ionomycin for 24 h, and then incubated with 100 nM Baf A1 for 4 h. The immunoprecipitated Myc-Ids was subjected to anti-K63-ubiquitin western blot. b Id-1 and Id-2 undergoes K63 ubiquitination. HEK293T cells were transiently transfected with HA-Ubi or HA-Ubi-K63R plasmids, treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then incubated with 100 nM Baf A1 for 4 h. The immunoprecipitated Myc-Id-1/2 was subjected to anti-HA western blot. c TRAF-6 is required for the K63-ubiquitination of Id-1/2. HEK293T cells were transfected with the indicated plasmids for 24 h, incubated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then treated with Baf A1 for 4 h. The immunoprecipitated Myc-Id-1/2 was subjected to anti-HA western blot. d TRAF-6 is required for the degradation of Id-1/2. HEK293T cells were transfected for 24 h with the negative control (NC) or TRAF-6 shRNA and then incubated for 24 h with 6 μM ionomycin or 100 nM EB1089. The cell lysates were then analyzed by western blot. e The bHLH domain of Id-1 was required for its interaction with TRAF-6. HEK293T cells were transiently transfected with the indicated plasmids for 24 h and then treated with 6 μM ionomycin for 24 h and 100 nM Baf A1 for 2 h before lysis. The whole-cell extracts were subjected to western blotting using the indicated antibodies after immunoprecipitation. f The lysine88 residues in the bHLH domain of Id-1 is more conserved during evolution (indicated with a red background and the other two with a blue background). g The K88 residue of Id-1 was necessary to the K63-linked ubiquitination of Id-1 induced by ionomycin. SK-N-SH cells were transiently co-transfected with plasmids encoding Myc-Id-1-WT or K88R and plasmid with HA-Ubiquitin, then treated with 6 μM ionomycin for 24 h, and with Baf A1 for 4 h. The cell lysates were then analyzed by western blot after the immunoprecipitation

    Journal: Nature Communications

    Article Title: CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

    doi: 10.1038/s41467-017-01272-2

    Figure Lengend Snippet: TRAF-6 induced the K63-linked ubiquitination of Id-1 at the K88 residue. a CaMKII and phosphorylation of Beclin 1 Ser90 are required for K63 ubiquitination of Ids. HEK293T cells were transiently co-transfected with the indicated plasmids, treated with 6 μM ionomycin for 24 h, and then incubated with 100 nM Baf A1 for 4 h. The immunoprecipitated Myc-Ids was subjected to anti-K63-ubiquitin western blot. b Id-1 and Id-2 undergoes K63 ubiquitination. HEK293T cells were transiently transfected with HA-Ubi or HA-Ubi-K63R plasmids, treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then incubated with 100 nM Baf A1 for 4 h. The immunoprecipitated Myc-Id-1/2 was subjected to anti-HA western blot. c TRAF-6 is required for the K63-ubiquitination of Id-1/2. HEK293T cells were transfected with the indicated plasmids for 24 h, incubated with 6 μM ionomycin or 100 nM EB1089 for 24 h, and then treated with Baf A1 for 4 h. The immunoprecipitated Myc-Id-1/2 was subjected to anti-HA western blot. d TRAF-6 is required for the degradation of Id-1/2. HEK293T cells were transfected for 24 h with the negative control (NC) or TRAF-6 shRNA and then incubated for 24 h with 6 μM ionomycin or 100 nM EB1089. The cell lysates were then analyzed by western blot. e The bHLH domain of Id-1 was required for its interaction with TRAF-6. HEK293T cells were transiently transfected with the indicated plasmids for 24 h and then treated with 6 μM ionomycin for 24 h and 100 nM Baf A1 for 2 h before lysis. The whole-cell extracts were subjected to western blotting using the indicated antibodies after immunoprecipitation. f The lysine88 residues in the bHLH domain of Id-1 is more conserved during evolution (indicated with a red background and the other two with a blue background). g The K88 residue of Id-1 was necessary to the K63-linked ubiquitination of Id-1 induced by ionomycin. SK-N-SH cells were transiently co-transfected with plasmids encoding Myc-Id-1-WT or K88R and plasmid with HA-Ubiquitin, then treated with 6 μM ionomycin for 24 h, and with Baf A1 for 4 h. The cell lysates were then analyzed by western blot after the immunoprecipitation

    Article Snippet: Ionomycin was purchased from Cell Signaling Technology.

    Techniques: Transfection, Incubation, Immunoprecipitation, Western Blot, Negative Control, shRNA, Lysis, Plasmid Preparation

    The phosphorylation of Ser36 is essential to the Lys-63-linked ubiquitylation of Id-1. a The interaction between Id-1 and CaMKII. HEK293T cells were transfected with the indicated plasmids for 24 h, incubated with 6 μM ionomycin or 10 μM KN-93 for 24 h. His-tagged proteins were precipitated and analyzed by western blot using the indicated antibodies. b The interaction between Id-1 and TRAF-6. HEK293T cells were transfected with the indicated plasmids for 24 h, and then treated with 6 μM ionomycin or 10 μM KN-93. The cells were treated with Baf A1 for 4 h before collect. The whole-cell lysates were analyzed by immunoblotting after precipitated by a Flag-M2 beads. c The illustration of Id-1 deletion constructs. d The 1–50 AAs fragment of Id-1 was essential to binding with CaMKII. HEK293 cells were co-transfected with vectors encoding CaMKII together with Myc-Id-1 FL (full-length) or its deletion mutants. Protein extracts were immunoprecipitated using anti-His antibody. e Tandem mass spectrometry analysis of Id-1 proteins after incubation with recombinant CaMKII in a kinase reaction. The data shown that the serine residue corresponding to Ser36 (indicated in red background in the peptide sequence) is phosphorylated. y, product ion numbered from C terminus of the peptide; b, product ion numbered from N terminus of the peptide. f The phosphorylation of Id-1 Ser36 was essential for the interaction of Id-1 and TRAF-6. HEK293T cells were transiently transfected with the indicated plasmids for 24 h and then treated with 6 μM ionomycin for 24 h and Baf A1 for 4 h before collect. The whole-cell extracts were subjected to western blotting using the indicated antibodies after an immunoprecipitation. g The phosphorylation of Id-1 Ser36 was required for the K63-ubiquitination of Id-1. HEK293T cells were transiently transfected with the indicated plasmids for 24 h and then treated with 6 μM ionomycin for 24 h and Baf A1 for 4 h before collect. The whole-cell extracts were subjected to western blotting using the indicated antibodies after an immunoprecipitation

    Journal: Nature Communications

    Article Title: CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

    doi: 10.1038/s41467-017-01272-2

    Figure Lengend Snippet: The phosphorylation of Ser36 is essential to the Lys-63-linked ubiquitylation of Id-1. a The interaction between Id-1 and CaMKII. HEK293T cells were transfected with the indicated plasmids for 24 h, incubated with 6 μM ionomycin or 10 μM KN-93 for 24 h. His-tagged proteins were precipitated and analyzed by western blot using the indicated antibodies. b The interaction between Id-1 and TRAF-6. HEK293T cells were transfected with the indicated plasmids for 24 h, and then treated with 6 μM ionomycin or 10 μM KN-93. The cells were treated with Baf A1 for 4 h before collect. The whole-cell lysates were analyzed by immunoblotting after precipitated by a Flag-M2 beads. c The illustration of Id-1 deletion constructs. d The 1–50 AAs fragment of Id-1 was essential to binding with CaMKII. HEK293 cells were co-transfected with vectors encoding CaMKII together with Myc-Id-1 FL (full-length) or its deletion mutants. Protein extracts were immunoprecipitated using anti-His antibody. e Tandem mass spectrometry analysis of Id-1 proteins after incubation with recombinant CaMKII in a kinase reaction. The data shown that the serine residue corresponding to Ser36 (indicated in red background in the peptide sequence) is phosphorylated. y, product ion numbered from C terminus of the peptide; b, product ion numbered from N terminus of the peptide. f The phosphorylation of Id-1 Ser36 was essential for the interaction of Id-1 and TRAF-6. HEK293T cells were transiently transfected with the indicated plasmids for 24 h and then treated with 6 μM ionomycin for 24 h and Baf A1 for 4 h before collect. The whole-cell extracts were subjected to western blotting using the indicated antibodies after an immunoprecipitation. g The phosphorylation of Id-1 Ser36 was required for the K63-ubiquitination of Id-1. HEK293T cells were transiently transfected with the indicated plasmids for 24 h and then treated with 6 μM ionomycin for 24 h and Baf A1 for 4 h before collect. The whole-cell extracts were subjected to western blotting using the indicated antibodies after an immunoprecipitation

    Article Snippet: Ionomycin was purchased from Cell Signaling Technology.

    Techniques: Transfection, Incubation, Western Blot, Construct, Atomic Absorption Spectroscopy, Binding Assay, Immunoprecipitation, Mass Spectrometry, Recombinant, Sequencing

    CaMKII activity affects the phosphorylation of Beclin 1 at Ser90. a Based on an analysis of the Beclin 1 amino acid sequence, Ser90 of Beclin 1 is strongly predicted to be a CaMKII phosphorylation site. b The predicted CaMKII phosphorylation sites in Beclin 1 are highly conserved during evolution. Putative CaMKII phosphorylation sites are indicated with a blue background. c The specificity of the anti-phospho-Ser90 Beclin 1 antibody. HEK293T cells were transiently transfected with plasmids encoding Flag-Beclin 1-WT, and the cells were incubated for 1 h at 30 °C in the absence or presence of ionomycin. The lysates of cells were incubated with or without λ-phosphatase. Flag-tagged proteins were immunoprecipitated and analyzed. d Ca (2+) mobilizing agents regulate Beclin 1 Ser90 phosphorylation. HEK293T cells were transiently transfected with the indicated plasmids for 48 h and then lysed, and Beclin 1 was precipitated using an anti-Flag antibody. The precipitates were analyzed by western blotting using an anti-phospho-Beclin 1-S90 antibody. e Ionomycin-induced phosphorylation of Beclin 1 on Ser90 occurs via a CaMKII-dependent pathway in a time-dependent and concentration-dependent manner. Both cell lines were treated with 6 μM ionomycin for the indicated periods or with various concentrations of ionomycin for 24 h. The cell extracts were analyzed by western blotting. f The EB1089-induced phosphorylation of Beclin 1 on Ser90 occurs via a CaMKII- dependent pathway in a time-dependent and concentration-dependent manner. Both cell lines were treated with 100 nM EB1089 for the indicated periods or various concentrations of EB1089 for 24 h. The cell extracts were analyzed by western blotting. g The inhibition of the phosphorylation of Beclin 1 by a CaMKII inhibitor. SK-N-SH cells were untreated or treated with 6 μM ionomycin, 100 nM EB1089, or 10 μM KN-93 for 24 h. The cell extracts were analyzed by western blotting

    Journal: Nature Communications

    Article Title: CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

    doi: 10.1038/s41467-017-01272-2

    Figure Lengend Snippet: CaMKII activity affects the phosphorylation of Beclin 1 at Ser90. a Based on an analysis of the Beclin 1 amino acid sequence, Ser90 of Beclin 1 is strongly predicted to be a CaMKII phosphorylation site. b The predicted CaMKII phosphorylation sites in Beclin 1 are highly conserved during evolution. Putative CaMKII phosphorylation sites are indicated with a blue background. c The specificity of the anti-phospho-Ser90 Beclin 1 antibody. HEK293T cells were transiently transfected with plasmids encoding Flag-Beclin 1-WT, and the cells were incubated for 1 h at 30 °C in the absence or presence of ionomycin. The lysates of cells were incubated with or without λ-phosphatase. Flag-tagged proteins were immunoprecipitated and analyzed. d Ca (2+) mobilizing agents regulate Beclin 1 Ser90 phosphorylation. HEK293T cells were transiently transfected with the indicated plasmids for 48 h and then lysed, and Beclin 1 was precipitated using an anti-Flag antibody. The precipitates were analyzed by western blotting using an anti-phospho-Beclin 1-S90 antibody. e Ionomycin-induced phosphorylation of Beclin 1 on Ser90 occurs via a CaMKII-dependent pathway in a time-dependent and concentration-dependent manner. Both cell lines were treated with 6 μM ionomycin for the indicated periods or with various concentrations of ionomycin for 24 h. The cell extracts were analyzed by western blotting. f The EB1089-induced phosphorylation of Beclin 1 on Ser90 occurs via a CaMKII- dependent pathway in a time-dependent and concentration-dependent manner. Both cell lines were treated with 100 nM EB1089 for the indicated periods or various concentrations of EB1089 for 24 h. The cell extracts were analyzed by western blotting. g The inhibition of the phosphorylation of Beclin 1 by a CaMKII inhibitor. SK-N-SH cells were untreated or treated with 6 μM ionomycin, 100 nM EB1089, or 10 μM KN-93 for 24 h. The cell extracts were analyzed by western blotting

    Article Snippet: Ionomycin was purchased from Cell Signaling Technology.

    Techniques: Activity Assay, Sequencing, Transfection, Incubation, Immunoprecipitation, Western Blot, Concentration Assay, Inhibition

    CaMKII directly phosphorylates Beclin 1 at Ser90. a The phosphorylation of Beclin 1-S90 requires activated CaMKII. SK-N-SH cells were transiently transfected with a control vector or plasmids encoding His-CaMKII-KD for 48 h. The whole-cell extracts were subjected to western blotting using the indicated antibodies. b CaMKII is required for phosphorylation of Beclin 1 on S90. HEK293T cells were transiently transfected with negative control or CaMKII shRNA, and then untreated or treated with 6 μM ionomycin or 100 nM EB1089 for 72 h. The cells were lysed and the indicated proteins were analyzed by western blot. c Increased association between CaMKII and Beclin 1 in cells treated with ionomycin or EB1089. HEK293T cells were treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, lysed, and then incubated with anti-CaMKII or control IgG antibodies prior to western blotting. The resulting immunoprecipitates (IP) were subjected to western blot analysis. TCL, the whole-cell lysate. d The association between His-tagged CaMKII and Flag-tagged Beclin 1. HEK293T cells or SK-N-SH cells were transiently co-transfected with plasmids encoding Flag-Beclin 1-WT and His-CaMKII-WT, lysed, and incubated with an anti-Flag antibody prior to western blot. The resulting immunoprecipitates were subjected to western blot analysis. TCL, the whole-cell lysate. e Phosphorylation of Beclin 1 by CaMKII upon ionomycin or EB1089 treatment. HEK293T cells were transiently transfected with plasmids encoding Flag-Beclin 1-WT, and the cells were incubated in 6 μM ionomycin or 100 nM EB1089. Flag-tagged proteins were precipitated and analyzed by western blot using an anti-phospho-S90-Beclin 1 and other indicated antibodies. f The phosphorylation-dependent activity of CaMKII on Beclin 1. HEK293T cells transfected with His-CaMKII-WT were untreated or treated with 6 μM ionomycin or 10 μM KN-93 for 24 h, then lysed and incubated with an anti-His antibody. Purified Beclin 1 fusion proteins were incubated with the immunoprecipitate, and in vitro kinase assays were performed. g In vitro phosphorylation of Beclin 1. HEK293T cells transfected with His-CaMKII-WT were treated with 6 μM ionomycin, then lysed and incubated with an anti-His antibody. Purified Beclin 1-WT and Beclin 1-S90A fusion proteins were incubated with the immunoprecipitate, and in vitro kinase assays were performed

    Journal: Nature Communications

    Article Title: CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

    doi: 10.1038/s41467-017-01272-2

    Figure Lengend Snippet: CaMKII directly phosphorylates Beclin 1 at Ser90. a The phosphorylation of Beclin 1-S90 requires activated CaMKII. SK-N-SH cells were transiently transfected with a control vector or plasmids encoding His-CaMKII-KD for 48 h. The whole-cell extracts were subjected to western blotting using the indicated antibodies. b CaMKII is required for phosphorylation of Beclin 1 on S90. HEK293T cells were transiently transfected with negative control or CaMKII shRNA, and then untreated or treated with 6 μM ionomycin or 100 nM EB1089 for 72 h. The cells were lysed and the indicated proteins were analyzed by western blot. c Increased association between CaMKII and Beclin 1 in cells treated with ionomycin or EB1089. HEK293T cells were treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, lysed, and then incubated with anti-CaMKII or control IgG antibodies prior to western blotting. The resulting immunoprecipitates (IP) were subjected to western blot analysis. TCL, the whole-cell lysate. d The association between His-tagged CaMKII and Flag-tagged Beclin 1. HEK293T cells or SK-N-SH cells were transiently co-transfected with plasmids encoding Flag-Beclin 1-WT and His-CaMKII-WT, lysed, and incubated with an anti-Flag antibody prior to western blot. The resulting immunoprecipitates were subjected to western blot analysis. TCL, the whole-cell lysate. e Phosphorylation of Beclin 1 by CaMKII upon ionomycin or EB1089 treatment. HEK293T cells were transiently transfected with plasmids encoding Flag-Beclin 1-WT, and the cells were incubated in 6 μM ionomycin or 100 nM EB1089. Flag-tagged proteins were precipitated and analyzed by western blot using an anti-phospho-S90-Beclin 1 and other indicated antibodies. f The phosphorylation-dependent activity of CaMKII on Beclin 1. HEK293T cells transfected with His-CaMKII-WT were untreated or treated with 6 μM ionomycin or 10 μM KN-93 for 24 h, then lysed and incubated with an anti-His antibody. Purified Beclin 1 fusion proteins were incubated with the immunoprecipitate, and in vitro kinase assays were performed. g In vitro phosphorylation of Beclin 1. HEK293T cells transfected with His-CaMKII-WT were treated with 6 μM ionomycin, then lysed and incubated with an anti-His antibody. Purified Beclin 1-WT and Beclin 1-S90A fusion proteins were incubated with the immunoprecipitate, and in vitro kinase assays were performed

    Article Snippet: Ionomycin was purchased from Cell Signaling Technology.

    Techniques: Transfection, Plasmid Preparation, Western Blot, Negative Control, shRNA, Incubation, Activity Assay, Purification, In Vitro

    Autophagy induced by CaMKII promotes degradation of inhibitor of differentiation proteins. a The degradation of Id-1 and Id-2 after ionomycin and EB1089 treatment. SK-N-SH cells were treated with 6 μM ionomycin or 100 nM EB1089 for the indicated periods or various concentrations of ionomycin or EB1089 for 24 h. The whole-cell lysates were analyzed by immunoblotting. b The ionomycin- and EB1089-induced degradation of Id-1 and Id-2 does not occur via the proteasome pathway. SK-N-SH cells were untreated or treated with 6 μM ionomycin or 100 nM EB1089 for 24 h and then incubated for 4 h in the presence or absence of 10 μM MG132. The total cell extracts were subjected to western blot using the indicated antibodies. c Autophagy is involved in the ionomycin-/EB1089-induced degradation of Id-1 and Id-2. SK-N-SH cells were untreated or treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, then incubated for 2 h in the presence or absence of 10 μM chloroquine (CQ). The whole-cell extracts were subjected to western blotting using the indicated antibodies. d Id-1/2 proteins are degraded via the autophagy pathway. SK-N-SH cells were transfected for 24 h with a negative control (NC) or Atg5 shRNA and then incubated for 24 h with 6 μM ionomycin or 100 nM EB1089. The cell lysates were then analyzed by western blotting. e The phosphorylation of Beclin 1 Ser90 is essential for the ionomycin-/EB1089-induced degradation of Id-1 and Id-2. HEK293T cells were transiently transfected with the indicated plasmids for 24 h and then treated with 6 μM ionomycin or 100 nM EB1089 for 24 h. The whole-cell extracts were subjected to western blotting using the indicated antibodies. f CaMKII is required for the degradation of Id-1 and Id-2 after ionomycin and EB1089 treatment. HEK293T cells were transiently transfected with negative control or CaMKII shRNA for 24 h and then treated with 6 μM ionomycin or 100 nM EB1089. The whole-cell extracts were subjected to western blotting using the indicated antibodies

    Journal: Nature Communications

    Article Title: CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

    doi: 10.1038/s41467-017-01272-2

    Figure Lengend Snippet: Autophagy induced by CaMKII promotes degradation of inhibitor of differentiation proteins. a The degradation of Id-1 and Id-2 after ionomycin and EB1089 treatment. SK-N-SH cells were treated with 6 μM ionomycin or 100 nM EB1089 for the indicated periods or various concentrations of ionomycin or EB1089 for 24 h. The whole-cell lysates were analyzed by immunoblotting. b The ionomycin- and EB1089-induced degradation of Id-1 and Id-2 does not occur via the proteasome pathway. SK-N-SH cells were untreated or treated with 6 μM ionomycin or 100 nM EB1089 for 24 h and then incubated for 4 h in the presence or absence of 10 μM MG132. The total cell extracts were subjected to western blot using the indicated antibodies. c Autophagy is involved in the ionomycin-/EB1089-induced degradation of Id-1 and Id-2. SK-N-SH cells were untreated or treated with 6 μM ionomycin or 100 nM EB1089 for 24 h, then incubated for 2 h in the presence or absence of 10 μM chloroquine (CQ). The whole-cell extracts were subjected to western blotting using the indicated antibodies. d Id-1/2 proteins are degraded via the autophagy pathway. SK-N-SH cells were transfected for 24 h with a negative control (NC) or Atg5 shRNA and then incubated for 24 h with 6 μM ionomycin or 100 nM EB1089. The cell lysates were then analyzed by western blotting. e The phosphorylation of Beclin 1 Ser90 is essential for the ionomycin-/EB1089-induced degradation of Id-1 and Id-2. HEK293T cells were transiently transfected with the indicated plasmids for 24 h and then treated with 6 μM ionomycin or 100 nM EB1089 for 24 h. The whole-cell extracts were subjected to western blotting using the indicated antibodies. f CaMKII is required for the degradation of Id-1 and Id-2 after ionomycin and EB1089 treatment. HEK293T cells were transiently transfected with negative control or CaMKII shRNA for 24 h and then treated with 6 μM ionomycin or 100 nM EB1089. The whole-cell extracts were subjected to western blotting using the indicated antibodies

    Article Snippet: Ionomycin was purchased from Cell Signaling Technology.

    Techniques: Incubation, Western Blot, Transfection, Negative Control, shRNA

    CaMKII induces autophagy through phosphorylation of Beclin 1 at Ser90 and the subsequent ubiquitination at Lys117. a Dissociation of the Bcl-2-Beclin 1 Complex. SK-N-SH cells treated with 6 μM ionomycin or 100 nM EB1089 were lysed and incubated with anti-Beclin 1 or normal mouse IgG antibodies prior to western blot analysis. The resulting immunoprecipitates were subjected to western blot analysis using the indicated antibodies. TCL, whole-cell lysate. b Autophagy induced by ionomycin in neuroblastoma cells. Both cell lines were treated with 6 μM ionomycin for the indicated periods or various concentrations of ionomycin for 24 h. The total cell lysates were analyzed for LC3 lipidation by immunoblotting. c LC3 mRNA expression was unaffected in cells treated with ionomycin or EB1089. The SK-N-SH cells were untreated or were treated with ionomycin or EB1089 of indicated concentration for 24 h. The expression level of LC3 mRNA was detected by real-time RT-PCR. The error bars represent the standard deviations (SD) calculated from three parallel experiments. d LC3 lipidation and p62 degradation in CaMKII-KD-expressing MEFs cells or control cells (Ctrl). MEFs cells transfected with a control vector or plasmids encoding His-CaMKII-KD were treated with 6 μM ionomycin for 24 h, then incubated for 2 h in the presence or absence of 100 nM Baf A1. The whole-cell extracts were subjected to western blot using the indicated antibodies. e LC3 lipidation and p62 degradation in CaMKII shRNA MEFs cells or negative control cells (NC). MEFs cells transfected with negative control or CaMKII shRNA were treated with 6 μM ionomycin for 24 h, then incubated for 2 h in the presence or absence of 100 nM Baf A1. The whole-cell extracts were subjected to western blot using the indicated antibodies. f LC3 lipidation in Beclin 1-deficient MEFs expressing WT Beclin 1 or S90A-Beclin 1. The cells were incubated in 6 μM ionomycin for 24 h, and total cell lysates were analyzed for LC3 lipidation by means of immunoblotting. g Autophagosome formation in MEFs deficient for Beclin 1. The cells were incubated in 6 μM ionomycin for 24 h and then fixed. LC3-positive puncta were shown as the means ± SEM of five random areas. ** P

    Journal: Nature Communications

    Article Title: CaMKII-mediated Beclin 1 phosphorylation regulates autophagy that promotes degradation of Id and neuroblastoma cell differentiation

    doi: 10.1038/s41467-017-01272-2

    Figure Lengend Snippet: CaMKII induces autophagy through phosphorylation of Beclin 1 at Ser90 and the subsequent ubiquitination at Lys117. a Dissociation of the Bcl-2-Beclin 1 Complex. SK-N-SH cells treated with 6 μM ionomycin or 100 nM EB1089 were lysed and incubated with anti-Beclin 1 or normal mouse IgG antibodies prior to western blot analysis. The resulting immunoprecipitates were subjected to western blot analysis using the indicated antibodies. TCL, whole-cell lysate. b Autophagy induced by ionomycin in neuroblastoma cells. Both cell lines were treated with 6 μM ionomycin for the indicated periods or various concentrations of ionomycin for 24 h. The total cell lysates were analyzed for LC3 lipidation by immunoblotting. c LC3 mRNA expression was unaffected in cells treated with ionomycin or EB1089. The SK-N-SH cells were untreated or were treated with ionomycin or EB1089 of indicated concentration for 24 h. The expression level of LC3 mRNA was detected by real-time RT-PCR. The error bars represent the standard deviations (SD) calculated from three parallel experiments. d LC3 lipidation and p62 degradation in CaMKII-KD-expressing MEFs cells or control cells (Ctrl). MEFs cells transfected with a control vector or plasmids encoding His-CaMKII-KD were treated with 6 μM ionomycin for 24 h, then incubated for 2 h in the presence or absence of 100 nM Baf A1. The whole-cell extracts were subjected to western blot using the indicated antibodies. e LC3 lipidation and p62 degradation in CaMKII shRNA MEFs cells or negative control cells (NC). MEFs cells transfected with negative control or CaMKII shRNA were treated with 6 μM ionomycin for 24 h, then incubated for 2 h in the presence or absence of 100 nM Baf A1. The whole-cell extracts were subjected to western blot using the indicated antibodies. f LC3 lipidation in Beclin 1-deficient MEFs expressing WT Beclin 1 or S90A-Beclin 1. The cells were incubated in 6 μM ionomycin for 24 h, and total cell lysates were analyzed for LC3 lipidation by means of immunoblotting. g Autophagosome formation in MEFs deficient for Beclin 1. The cells were incubated in 6 μM ionomycin for 24 h and then fixed. LC3-positive puncta were shown as the means ± SEM of five random areas. ** P

    Article Snippet: Ionomycin was purchased from Cell Signaling Technology.

    Techniques: Incubation, Western Blot, Expressing, Concentration Assay, Quantitative RT-PCR, Transfection, Plasmid Preparation, shRNA, Negative Control

    Percentage of sample determined to be electroporated (EP) with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant differences in the percent of EP cells. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be electroporated (EP) with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant differences in the percent of EP cells. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be EP with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percentage of EP cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be EP with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percentage of EP cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be RE with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percent of RE cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be RE with a 2.06 kV/cm PEF intensity. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. Only 40- μ s pulses resulted in significant differences in the percent of RE cells. Error bars represent a 95% confidence interval. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be RE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant increase in the percent of RE cells in the treatment samples, whereas at 6.0 and 6.9 kV/cm, we found a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be RE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 1.8 and 2.0 kV/cm resulted in significant increase in the percent of RE cells in the treatment samples, whereas at 6.0 and 6.9 kV/cm, we found a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be IRE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 6.0 and 6.9 kV/cm resulted in a statistically significant increase in IRE in the treatment samples. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be IRE with a 40- μ s pulse duration. The control group was EP in HBSS, and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 6.0 and 6.9 kV/cm resulted in a statistically significant increase in IRE in the treatment samples. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    Percentage of sample determined to be RE with a 100- μ s pulse duration. The control group was EP in HBSS and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 4.0 kV/cm had a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Journal: Biophysical Journal

    Article Title: Ionomycin-Induced Changes in Membrane Potential Alter Electroporation Outcomes in HL-60 Cells

    doi: 10.1016/j.bpj.2018.05.018

    Figure Lengend Snippet: Percentage of sample determined to be RE with a 100- μ s pulse duration. The control group was EP in HBSS and the treatment group was EP in HBSS supplemented with 100 nM ionomycin. The average values of each sampled PEF intensity are depicted by “o” for control and “ ∗ ” for treatment. The error bars represent a 95% confidence interval. PEF intensities of 4.0 kV/cm had a statistically significant reduction in RE in the treatment samples. Regression lines represent the fitted cubic splines.

    Article Snippet: An equal quantity of 60 μ M PI solution (Fisher Scientific International, Pittsburgh, PA) (dissolved in HBSS) was added to the cell/HBSS mixture, resulting in a final PI concentration of 30 μ M. For the treatment group (HBSS + ionomycin experiments), the control solution (PI + HBSS) was supplemented with 100 nM ionomycin (Fisher Scientific).

    Techniques:

    BSH effect on intracellular markers of systemic NK cells. Following NK cell enrichment, NK cells were stimulated with PMA/Ionomycin and blocked with Brefeldin A for 4hrs. The differences of day2 or day21 and day-1 are shown. Data are presented as mean±std.dev. N = 9–14. Data are shown as whiskers with 10–90 percentiles. *significantly different (p = 0.049), tested with two sample t test.

    Journal: PLoS ONE

    Article Title: Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study

    doi: 10.1371/journal.pone.0147742

    Figure Lengend Snippet: BSH effect on intracellular markers of systemic NK cells. Following NK cell enrichment, NK cells were stimulated with PMA/Ionomycin and blocked with Brefeldin A for 4hrs. The differences of day2 or day21 and day-1 are shown. Data are presented as mean±std.dev. N = 9–14. Data are shown as whiskers with 10–90 percentiles. *significantly different (p = 0.049), tested with two sample t test.

    Article Snippet: NK cell treatment NK cells were either analyzed directly after the enrichment or were stimulated for 4hrs with 50ng/ml phorbol 12-myristate 13-acetate (PMA; Acros Organics, Fisher Scientific) and 1μg/ml ionomycin (MP Biomedicals).

    Techniques:

    Overview of sample collection and processing. (A) Study design and sample collection. Details of the complete study have been published previously [ 1 ]. (B) Blood samples were stained for total leukocyte populations or used for NK cell enrichment. NK cells were analyzed for surface marker expression or cytokine production either naive or stimulated with PMA and ionomycin (Iono). Half of the peripheral blood mononuclear cells (PBMCs) were frozen and used later for the cytotoxicity assay.

    Journal: PLoS ONE

    Article Title: Effect of Broccoli Sprouts and Live Attenuated Influenza Virus on Peripheral Blood Natural Killer Cells: A Randomized, Double-Blind Study

    doi: 10.1371/journal.pone.0147742

    Figure Lengend Snippet: Overview of sample collection and processing. (A) Study design and sample collection. Details of the complete study have been published previously [ 1 ]. (B) Blood samples were stained for total leukocyte populations or used for NK cell enrichment. NK cells were analyzed for surface marker expression or cytokine production either naive or stimulated with PMA and ionomycin (Iono). Half of the peripheral blood mononuclear cells (PBMCs) were frozen and used later for the cytotoxicity assay.

    Article Snippet: NK cell treatment NK cells were either analyzed directly after the enrichment or were stimulated for 4hrs with 50ng/ml phorbol 12-myristate 13-acetate (PMA; Acros Organics, Fisher Scientific) and 1μg/ml ionomycin (MP Biomedicals).

    Techniques: Staining, Marker, Expressing, Cytotoxicity Assay

    Characterization of aberrant T cells in LTAC mice. (A and B) Frequencies (left) and absolute numbers (right) of TCRβ + cells of CD45 + cells from mLNs (A) or cLP (B) of WT (n = 9–10, filled circle) and LTAC mice (n = 9–11, open circle) determined by flow cytometry analysis. Horizontal bars represent mean. (C) (Left and middle plots) Flow cytometry of CD4 + and CD8α + cell subsets in TCRβ + cells. (Right panels) Frequencies of CD4 + cell subset in TCRβ + cells. The plots are representative of at least three independent experiments. mLNs (upper) or cLP (lower) of WT (n = 7–10, filled circle) and LTAC mice (n = 8–10, open circle), determined by flow cytometry analysis. Horizontal bars represent mean. (D) Genomic DNAs from the sorted T cells in each tissue indicated were subjected to PCR amplification to detect flox (= TAK1 WT) or Δ (= TAK1 KO) allele in the TAK1 genomic locus. The DNA size difference between the flox and Δ bands is shown. Specific DNAs for Cre, loaded in each well, were also shown to prove its existence in the genome. (E) Flow cytometry of intracellular cytokines in TCRβ + CD4 + cells from cLP after culture with PMA + Ionomycin for 5 hours in the presence of GolgiStop. The plots are representative of four independent experiments. (F) Frequencies of each subset of cytokine-producing cells in TCRβ + CD4 + cells in cLP of WT (n = 4, filled circle) and LTAC mice (n = 4, open circle), calculated by flow cytometry analysis from (E). Horizontal bars represent mean. (G) Flow cytometry of gut homing receptors by mLN TCRβ + CD4 + cells. The histograms are representative of at least three independent experiments. (H) Frequencies of α4β7 hi , CCR9 + and CD103 + cells of TCRβ + CD4 + cells in the mLNs of WT (n ≧ 5, filled circle) and LTAC mice (n ≧ 5, open circle), determined by flow cytometry analysis from (H). Horizontal bars represent mean. In (A), (B), (C), (F) and (H), unpaired t tests were performed. Statistical significance was indicated by * P

    Journal: PLoS ONE

    Article Title: Conditional Deletion of TAK1 in T Cells Reveals a Pivotal Role of TCRαβ+ Intraepithelial Lymphocytes in Preventing Lymphopenia-Associated Colitis

    doi: 10.1371/journal.pone.0128761

    Figure Lengend Snippet: Characterization of aberrant T cells in LTAC mice. (A and B) Frequencies (left) and absolute numbers (right) of TCRβ + cells of CD45 + cells from mLNs (A) or cLP (B) of WT (n = 9–10, filled circle) and LTAC mice (n = 9–11, open circle) determined by flow cytometry analysis. Horizontal bars represent mean. (C) (Left and middle plots) Flow cytometry of CD4 + and CD8α + cell subsets in TCRβ + cells. (Right panels) Frequencies of CD4 + cell subset in TCRβ + cells. The plots are representative of at least three independent experiments. mLNs (upper) or cLP (lower) of WT (n = 7–10, filled circle) and LTAC mice (n = 8–10, open circle), determined by flow cytometry analysis. Horizontal bars represent mean. (D) Genomic DNAs from the sorted T cells in each tissue indicated were subjected to PCR amplification to detect flox (= TAK1 WT) or Δ (= TAK1 KO) allele in the TAK1 genomic locus. The DNA size difference between the flox and Δ bands is shown. Specific DNAs for Cre, loaded in each well, were also shown to prove its existence in the genome. (E) Flow cytometry of intracellular cytokines in TCRβ + CD4 + cells from cLP after culture with PMA + Ionomycin for 5 hours in the presence of GolgiStop. The plots are representative of four independent experiments. (F) Frequencies of each subset of cytokine-producing cells in TCRβ + CD4 + cells in cLP of WT (n = 4, filled circle) and LTAC mice (n = 4, open circle), calculated by flow cytometry analysis from (E). Horizontal bars represent mean. (G) Flow cytometry of gut homing receptors by mLN TCRβ + CD4 + cells. The histograms are representative of at least three independent experiments. (H) Frequencies of α4β7 hi , CCR9 + and CD103 + cells of TCRβ + CD4 + cells in the mLNs of WT (n ≧ 5, filled circle) and LTAC mice (n ≧ 5, open circle), determined by flow cytometry analysis from (H). Horizontal bars represent mean. In (A), (B), (C), (F) and (H), unpaired t tests were performed. Statistical significance was indicated by * P

    Article Snippet: For intracellular cytokine staining, cells were cultured with 50 ng/ml phorbol 12-myristate 13-acetate (PMA) (Wako) plus 500 ng/ml ionomycin (Wako) for 5 hours in the presence of GolgiStop (BD Bioscience).

    Techniques: Mouse Assay, Flow Cytometry, Cytometry, Polymerase Chain Reaction, Amplification

    Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM ionomycin (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.

    Journal: Scientific Reports

    Article Title: Mitochondrial behaviour throughout the lytic cycle of Toxoplasma gondii

    doi: 10.1038/srep42746

    Figure Lengend Snippet: Mitochondrial morphology of T. gondii tachyzoites changes upon host cell egress. (a) Fluorescence micrograph of intracellular (In) and extracellular (out) populations of T. gondii tachyzoites taken utilizing the signal from 215430-YFP (green in the merge with the brightfield, top panels, and in the bottom panels). Bars 5 μm. (b) Three representative images of each of the observed shapes of mitochondria in extracellular parasites and their classification. Each example shows the fluorescent signal image on the left and the merge of fluorescence and brightfield on the right. The color-coding of the frame (lasso – green; sperm-like – orange; collapsed – red) is maintained throughout all the figures. (c) Proportions of the morphologies scored in intracellular parasites (Intracellular, 776 parasites, over 6 independent experiments); in parasites mechanically released from host cell into full growth medium (Full, 864 parasites, over 9 independent experiments) or into diluted growth medium (12%, 653 parasites, over 2 independent experiments) immediately after release; and in parasites induced to egress by 2 μM ionomycin (Ionomycin, 1177 parasites, over 6 independent experiments) immediately after release. Error bars are standard deviation. (d) Super-resolution microscopy images of the mitochondrial lasso morphology in intracellular (left) and the three main mitochondrial morphologies observed in extracellular: lasso, sperm-like and collapsed (right) shown as projection of all Z stacks (top) and as 3D reconstruction (bottom). 215430 - green. DAPI - blue. Bar 2 μm.

    Article Snippet: Images were taken every 10 seconds for a total of 5 minutes and 2 μM ionomycin (Santa Cruz Biotechnology) was added after 2–4 time points were imaged.

    Techniques: Fluorescence, Standard Deviation, Microscopy

    Effect of MFN2 on the immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Effect of MFN2 on the immune function of Jurkat cells in response to PMA/ionomycin. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmol/L) for various lengths of time (6, 12, 24, and 48 h). (a) A methyl-thiazolyl-tetrazolium cell proliferation assay was used to assess Jurkat cells activity. Levels of IL-2 (b) and the IFN-γ/IL-4 ratio (c) were measured by ELISA. Results are shown as mean ± standard deviation. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Transfection, Proliferation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Regulation of calcineurin expression reverses MFN2-induced changes in immune function of Jurkat cells. After being transfected with MFN2-RNAi followed by LV-calcineurin, Jurkat cells were treated with PMA plus ionomycin for 24 h. Then, cell proliferative activity (a), IL-2 levels (c), and the ratio of IFN-γ/IL-4 (e) were assessed. Jurkat cells were pretreated LV-MFN2 followed by FK-506 and then stimulated with PMA plus ionomycin for 24 h. The cell proliferative activity (b), IL-2 levels (d), and the ratio of IFN-γ/IL-4 (f) were detected. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Regulation of calcineurin expression reverses MFN2-induced changes in immune function of Jurkat cells. After being transfected with MFN2-RNAi followed by LV-calcineurin, Jurkat cells were treated with PMA plus ionomycin for 24 h. Then, cell proliferative activity (a), IL-2 levels (c), and the ratio of IFN-γ/IL-4 (e) were assessed. Jurkat cells were pretreated LV-MFN2 followed by FK-506 and then stimulated with PMA plus ionomycin for 24 h. The cell proliferative activity (b), IL-2 levels (d), and the ratio of IFN-γ/IL-4 (f) were detected. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Expressing, Transfection, Activity Assay

    Effect of MFN2 on intracellular calcium, calcineurin expression, and NFAT activity in Jurkat cells. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmmol/L) for various lengths of time. (a and c) Intracellular calcium was measured by FACS with the fluorescent probe Fluo-3/AM. (b) Calcineurin activity was measured using a calcineurin assay kit. (d) NFAT activity in Jurkat cells was measured by ELISA. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Effect of MFN2 on intracellular calcium, calcineurin expression, and NFAT activity in Jurkat cells. Jurkat cells were transfected with LV-MFN2 and MFN2-RNAi and then stimulated with PMA (50 ng/ml) plus ionomycin (1 mmmol/L) for various lengths of time. (a and c) Intracellular calcium was measured by FACS with the fluorescent probe Fluo-3/AM. (b) Calcineurin activity was measured using a calcineurin assay kit. (d) NFAT activity in Jurkat cells was measured by ELISA. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Expressing, Activity Assay, Transfection, FACS, Enzyme-linked Immunosorbent Assay

    Regulation of calcineurin expression reverses MFN2-induced NFAT activation in Jurkat cells. After various pretreatments, Jurkat cells were stimulated with PMA plus ionomycin for 24 h. Jurkat cells were pretreated with MFN2-RNAi and then LV-calcineurin, and calcineurin activity was measured by commercial assay kit (a) and NFAT activity was determined by ELISA (c). Jurkat cells were pretreated with LV-MFN2 and then FK-506, and calcineurin activity (b) and NFAT activity (d) were measured. * P

    Journal: Chinese Medical Journal

    Article Title: Role of the Ca2+-Calcineurin-Nuclear Factor of Activated T cell Pathway in Mitofusin-2-Mediated Immune Function of Jurkat Cells

    doi: 10.4103/0366-6999.223855

    Figure Lengend Snippet: Regulation of calcineurin expression reverses MFN2-induced NFAT activation in Jurkat cells. After various pretreatments, Jurkat cells were stimulated with PMA plus ionomycin for 24 h. Jurkat cells were pretreated with MFN2-RNAi and then LV-calcineurin, and calcineurin activity was measured by commercial assay kit (a) and NFAT activity was determined by ELISA (c). Jurkat cells were pretreated with LV-MFN2 and then FK-506, and calcineurin activity (b) and NFAT activity (d) were measured. * P

    Article Snippet: Phorbol myristate acetate (PMA) and ionomycin were purchased from the Beyotime Institute (Nanjing, China).

    Techniques: Expressing, Activation Assay, Activity Assay, Enzyme-linked Immunosorbent Assay

    T cell development and stress-induced apoptosis are normal in A1 −/− mice. ( a , left) Representative FACS plots of developing T cells in the thymus of wild-type and A1 −/− mice. T cells develop in the thymus from the CD4/CD8 double-negative (DN) stage, becoming double positive (DP) and then differentiating into either CD4 or CD8 single-positive cells. The DN population is further subdivided into DN1–4 phases: DN1 (CD44 + CD25 − ), DN2 (CD44 + CD25 + ), DN3 (CD44 − CD25 + ) and DN4 (CD44 − CD25 − ). ( a , right) Quantified total cell numbers for developing T cell populations in wild-type and A1 −/− mice. ( b ) Thymocytes harvested from wild-type and A1 −/− mice were put into culture with the following apoptosis-inducing compounds: ABT-737 (737) 0.5 μ M, dexamethasone (Dexa) 0.5 ng/ml, etoposide (Etopo) 0.5 μ g/ml, ionomycin (Iono), 0.1 μ g/ml, PMA 0.5 ng/ml or DMSO as a control. Apoptosis was assessed by FACS analysis after staining with fluorochrome-conjugated Annexin-V and PI (5 μ g/ml) at 24 h post treatment. Data are representative of six mice for each genotype, with each treatment performed in triplicate

    Journal: Cell Death and Differentiation

    Article Title: Characterisation of mice lacking all functional isoforms of the pro-survival BCL-2 family member A1 reveals minor defects in the haematopoietic compartment

    doi: 10.1038/cdd.2016.156

    Figure Lengend Snippet: T cell development and stress-induced apoptosis are normal in A1 −/− mice. ( a , left) Representative FACS plots of developing T cells in the thymus of wild-type and A1 −/− mice. T cells develop in the thymus from the CD4/CD8 double-negative (DN) stage, becoming double positive (DP) and then differentiating into either CD4 or CD8 single-positive cells. The DN population is further subdivided into DN1–4 phases: DN1 (CD44 + CD25 − ), DN2 (CD44 + CD25 + ), DN3 (CD44 − CD25 + ) and DN4 (CD44 − CD25 − ). ( a , right) Quantified total cell numbers for developing T cell populations in wild-type and A1 −/− mice. ( b ) Thymocytes harvested from wild-type and A1 −/− mice were put into culture with the following apoptosis-inducing compounds: ABT-737 (737) 0.5 μ M, dexamethasone (Dexa) 0.5 ng/ml, etoposide (Etopo) 0.5 μ g/ml, ionomycin (Iono), 0.1 μ g/ml, PMA 0.5 ng/ml or DMSO as a control. Apoptosis was assessed by FACS analysis after staining with fluorochrome-conjugated Annexin-V and PI (5 μ g/ml) at 24 h post treatment. Data are representative of six mice for each genotype, with each treatment performed in triplicate

    Article Snippet: For ionomycin, LPS and IL-33 treatment survival assays, cells were washed to remove cytokines and then treated with 1 μ g/ml ionomycin, 5 μ g/ml ultrapure LPS ( E. coli 0111:B4 strain–TLR4 ligand, InvivoGen, San Diego, CA, USA), 10 ng/ml IL-33 (produced in-house and kindly provided by Dr. Ajithkumar Vasanthakumar of WEHI) or medium alone (no cytokines added).

    Techniques: Mouse Assay, FACS, Staining

    LMP-induced increases in cytosolic [Ca 2+ ]: kinetics and attenuation by high-level LMP. A , B , and D : changes in cytosolic [Ca 2+ ] were measured in fluo 4-loaded BMDCs (WT or Casp1 −/− as indicated) primed with LPS. Baseline fluo 4 fluorescence was recorded for 5 min before stimulation of the cells with the indicated concentrations of LLME or 6 μM ionomycin. D : parallel wells of BMDCs were treated with or without 100 μM CA074Me during the final 2 h of the 4-h LPS priming incubation. In A and B , cytosolic [Ca 2+ ] was calculated based on calibration after Triton X-100 (Tx)-mediated release of intracellular fluo 4 into the 1.5 mM CaCl 2 -containing medium followed by chelation of Ca 2+ with EGTA; data points represent the means ± SE from 4 experiments. In D , time-dependent changes in fluo 4 fluorescence for WT BMDCs are directly plotted; data represent the average ± range of technical duplicates from a single experiment. C : plot of the differential rates of [Ca 2+ ] increase measured between the 10- and 20-min time points from the time courses in A and B ; ** P

    Journal: American Journal of Physiology - Cell Physiology

    Article Title: NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx

    doi: 10.1152/ajpcell.00298.2015

    Figure Lengend Snippet: LMP-induced increases in cytosolic [Ca 2+ ]: kinetics and attenuation by high-level LMP. A , B , and D : changes in cytosolic [Ca 2+ ] were measured in fluo 4-loaded BMDCs (WT or Casp1 −/− as indicated) primed with LPS. Baseline fluo 4 fluorescence was recorded for 5 min before stimulation of the cells with the indicated concentrations of LLME or 6 μM ionomycin. D : parallel wells of BMDCs were treated with or without 100 μM CA074Me during the final 2 h of the 4-h LPS priming incubation. In A and B , cytosolic [Ca 2+ ] was calculated based on calibration after Triton X-100 (Tx)-mediated release of intracellular fluo 4 into the 1.5 mM CaCl 2 -containing medium followed by chelation of Ca 2+ with EGTA; data points represent the means ± SE from 4 experiments. In D , time-dependent changes in fluo 4 fluorescence for WT BMDCs are directly plotted; data represent the average ± range of technical duplicates from a single experiment. C : plot of the differential rates of [Ca 2+ ] increase measured between the 10- and 20-min time points from the time courses in A and B ; ** P

    Article Snippet: Key reagents and their sources were as follows: Escherichia coli LPS serotype O1101:B4 (List Biological Laboratories), nigericin (NG) (Sigma-Aldrich), PR-619 (Sigma-Aldrich), ionomycin (LC Laboratories), H-Leu-Leu-OMe·HBr (Bachem), CA-074-methyl ester (Bachem), Imject Alum (Pierce), monosodium urate (Invivogen), disuccinimidyl suberate (DSS; Sigma-Aldrich), anti-caspase-1 (p20) mouse mAb (Casper-1; Adipogen), anti-NLRP3 mouse mAb (Cryo-2; Adipogen), and anti-NLRP3 rat mAb (Clone 768319; R & D Systems).

    Techniques: Fluorescence, Incubation