Journal: Nature communications

Article Title: Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states.

doi: 10.1038/ncomms12057

Figure Lengend Snippet: Figure 3 | The refractory period is controlled downstream of TNFR and upstream of IKK. (a) Response to alternate TNFa and IL-1b pulses applied at 0 and 60 min Shown is the mean (±s.d.) of normalized total IkBa-GFP intensity in single C9 cells. Timing of TNFa (0min) and IL-1b (60min) stimulation represented with blue and pink bars, respectively. (b) Comparison between responses to two TNFa pulses (TT) and alternate TNFa and IL-1b (TI) pulses at 60min interval. Shown are fractions (mean ±s.d.) of C9 cells that responded to the second pulse. (c) NF-kB amplitude of cells responding to two TNFa pulses (TT) and alternate TNFa and IL-1b (TI) pulses at 60 min interval. Shown are the peak 2 (P2) p65-mCherry translocation amplitudes (expressed as the fraction of the peak 1 amplitude, P1) of individual C9L cells, with corresponding mean and±data range per condition. In TI, TNFa and IL-1b pulses applied at 0 and 60min, respectively. Statistical difference assessed with a Mann-Whitney test (**** P valo0.0001). (d) Immunoblotting analysis of IkBa and serine 536-phosphorylated NF-kB p65 levels in WTcells stimulated with two pulses of TNFa, or alternate TNFa and IL-1b pulses at 60 min interval. Timing of TNFa and IL-1b stimulation represented with blue and pink bars, respectively. (e) Confocal microscopy images of WT cells stimulated with two pulses of fluorescently labelled TNFa at 60min interval. FITC-conjugated TNFa (top) was applied at 0min and measured 10 min after stimulation. Tx-Red-conjugated TNFa (middle) was applied on the same cells at 60min, and measured at 70min after the start of the experiment. Corresponding bright field images shown at the bottom. Scale bar, 20mm. (f) TNFa internalization in WTcells as in e. Shown is total fluorescence levels per cell measured at 10min after stimulation at 0 and 60 min, respectively. (g) Flow cytometry analysis of TNFR1 receptor expression. WT SK-N-AS cells were stimulated with 5 min pulse of TNFa and TNFR1 expression was measured by flow cytometry at 5, 60 and 100min after treatment (in addition to untreated and unlabelled controls).

Article Snippet: Human recombinant TNFa biotin conjugate (1mgml 1, Fluorokine, R&D Systems, Wiesbaden) was diluted to 25 ngml 1 in either 20ml of avidin-FITC (10 mgml 1) or 2 ml avidin-Texas-Red (2mgml 1, Life Technologies) and made up to 50 ml with minimum essential medium.

Techniques: Comparison, Translocation Assay, MANN-WHITNEY, Western Blot, Confocal Microscopy, Flow Cytometry, Expressing, Cytometry

Journal: Nature communications

Article Title: Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states.

doi: 10.1038/ncomms12057

Figure Lengend Snippet: Figure 5 | Single-cell responses to pulsed TNFa stimulation are imprinted. (a) Schematic representation of the intrinsic (blue) and extrinsic (yellow) noise in the NF-kB system. (b) Different noise models fit single-cell data. Shown is the comparison between simulation (300 cells) of extrinsic noise (via the distributed A20 transcription rate) and intrinsic noise (via the stochastic regulation of A20 gene activity) model, and the data for TNFa pulses at 70 min interval. (c) Simulated single-cell traces (shown as NF-kB:IkBa complex levels, in different coloured lines) for different noise models. TNFa applied at two 70 min intervals separated by 4 h equilibration phase (as depicted with blue bars). (d) C9 cell responses to equilibrated TNFa pulses (as in c). Shown are means (in green, ±s.d.) of normalized single-cell total IkBa-GFP intensities in nine non-responsive (left panel) and 32 responsive cells (right panel) to second (p2) and fourth (p4) pulse. (e) Comparison between model simulations and the data for equilibrated TNFa pulses. Simulation performed with 300 cells assuming intrinsic (in yellow) and extrinsic (in blue) noise (as in c). Data from d presented in fraction of cells (total 79) responding to second ( þ p2, p4), fourth ( p2, þ p4) or both second and fourth ( þ p2, þ p4) pulses. (f) Principal component analysis of single-cell data from d. For each cell, 140 min sub-trajectories corresponding to two stimulation phases were considered (depicted with symbols connected with different colour lines). Responsive and non-responsive cell clusters outlined with dashed lines. (g) Daughter cell analysis in response to two TNFa pulses at 70 min interval. Time from cell division to stimulation recorded. (h) Representative daughter C9 cell responses (as in g): Cells (indicated with stars, IkBa-eGFP intensities shown) respond to the first (depicted at 10 min after stimulation), as well as to the second TNFa pulse (at 80 min after start of the experiment). Scale bar, 20 mm. (i) Representative daughter cells trajectories (from the experiment in g). (j) Fractions of homogenous and heterogeneous daughter cells responses. 56 pairs stimulated as in g, stratified by patterns of the IkBa-eGFP signal.

Article Snippet: Human recombinant TNFa biotin conjugate (1mgml 1, Fluorokine, R&D Systems, Wiesbaden) was diluted to 25 ngml 1 in either 20ml of avidin-FITC (10 mgml 1) or 2 ml avidin-Texas-Red (2mgml 1, Life Technologies) and made up to 50 ml with minimum essential medium.

Techniques: Comparison, Activity Assay, Cell Analysis

Journal: Nature communications

Article Title: Signal transduction controls heterogeneous NF-κB dynamics and target gene expression through cytokine-specific refractory states.

doi: 10.1038/ncomms12057

Figure Lengend Snippet: Figure 6 | Cells refractory to TNFa encode IL-1b signals. (a) Representative single C9L cell traces stimulated with three pulses of TNFa (TTT, depicted with blue bars) or alternate TNFa and IL-1b pulses (TIT, depicted with pink bars) at 50 min intervals. Shown are normalized total IkBa-eGFP fluorescent intensities and the nuclear to total (N/T) ratios of the p65-mCherry signal. Time depicted in minutes. (b) Nuclear NF-kB activity in cells stimulated with three pulses of TNFa (TTT) or alternate TNFa and IL-1b pulses (TIT) at 50 min interval. Shown is the area under the N/Tp65-mCherry trajectory (AUC) for cells as in a (base-line corrected trajectories were normalized to the first peak amplitude). (c) Schematic representation of the gene expression assay. Cells were stimulated with pulses of TNFa and IL-1b at different times (as indicated with blue and pink bars, respectively). Measurement obtained at 130 min after start of the experiment. (d) Heat map of gene expression levels for data from c. Clustering performed for log2 fold changes (as indicated with the colour scale) of three replicates. (e) Gene expression levels for IkBa, A20, CSF2, CXCL2 and IL8 transcript levels from data in d. Shown are mean expression levels (±s.d.) per condition, respectively. (f) Differential gene expression analysis between TNFa/IL-1b/TNFa (TIT) and TNFa/TNFa/TNFa (TTT) stimulation. Shown are log2 of expression fold changes for NF-kB system genes (A20, IkBa, IkBe, Rel, RelB, NF-kB1 and NF-kB2) and cytokine response genes (CXCL2, CXCL1, IL8, TNFAIP6, CSF2, TNFa, LIF, IL-1b and CCL2).

Article Snippet: Human recombinant TNFa biotin conjugate (1mgml 1, Fluorokine, R&D Systems, Wiesbaden) was diluted to 25 ngml 1 in either 20ml of avidin-FITC (10 mgml 1) or 2 ml avidin-Texas-Red (2mgml 1, Life Technologies) and made up to 50 ml with minimum essential medium.

Techniques: Activity Assay, Gene Expression, Expressing

Journal: Nutrients

Article Title: Construction and Evaluation of a Novel MAP Immunoassay for 9 Nutrition-and-Health-Related Protein Markers Based on Multiplex Liquid Protein Chip Technique

doi: 10.3390/nu15061522

Figure Lengend Snippet: Reagents used in this research.

Article Snippet: D-D detection antibody , NB100-73038B , Novus Biologicals, Inc. (Centennial, CO, USA).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Diagnostic Assay