Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A ) Overlay histograms of LFA-1 diffusion on monocytes (4578 trajectories), resting mDCs (26756 trajectories) and CCL21 activated mDCs (4213 trajectories). ( B ) Percentage of total mobile LFA-1 population (normalized to 100%) displaying slow and fast diffusion on monocytes, mDCs and 2 min CCL21 activated mDCs. ( C ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on monocytes, mDCs and 2 min CCL21 activated mDCs. ( D ) Stationary fraction of LFA-1 on monocytes, mDCs and CCL21 activated mDCs, displayed as the difference from the total stationary fraction on mDCs, which serve here as the default. Data from A–D on CCL21 activated mDCs is based on 22 cells in independent experiments from 3 different donors. ( E ) Percentage of the stationary, slow and fast diffusing LFA-1 molecules at different time points after CCL21 activation. ( F ) D values for the total mobile, and slow and fast fractions of LFA-1 at different time points after CCL21 activation. Data from E and F is based on 11 cells, 11 independent samples and around 2000 trajectories per time point. A – F Means ± SEM are depicted. The One-way ANOVA followed by the Tukey multiple comparison test were used to determine significant differences between means. The resulting P values are indicated as follows: ns (P >0.05); * (P<0.05) and *** (P<0.0001).

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Diffusion-based Assay, Activation Assay, Comparison

Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A,B ) ICAM-1 (either monomeric: ICAMm, or as nano-aggregates: ICAMagg) was added to resting mDCs and mobility was measured before, and between 1 and 5 min after addition. ( A ) Stationary fraction of LFA-1 molecules on resting mDCs and mDCs + either monomeric ICAM-1 or ICAM-1 nano-aggregates, displayed as the difference with respect to the total stationary fraction on resting mDCs, which serve here as the default. ( B ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on resting mDCs and after addition of either monomeric ICAM-1 or ICAM-1 nano-aggregates. 30 cells divided over 2 independent experiments (3393 trajectories) were imaged for the ICAMm condition and 10 cells (684 trajectories) for ICAMagg. ( C, D ) ICAM-1 (either monomeric or nano-aggregates) was added together with CCL21 to mDCs and mobility was measured before, and 2 minutes after addition. ( C ) Stationary fraction of LFA-1 molecules on resting mDCs (serving as reference control), CCL21 activated mDCs and CCL21 activated mDCs + either monomeric ICAM-1 or ICAM-1 nano-aggregates, displayed as the difference with respect to the total stationary fraction on resting mDCs. ( D ) Diffusion coefficient of the total mobile population, and slow and fast diffusing fractions of LFA-1 on resting mDCs, CCL21 activated mDCs and after simultaneous addition of CCL21 and either monomeric ICAM-1 or ICAM-1 nano-aggregates. 16 cells (8423 trajectories) from 2 different donors divided over 5 independent samples (ICAMm) and 7 cells (314 trajectories) from 2 different donors divided over 7 independent samples (ICAMagg) were imaged. ( E ) Quantification of fluorescent ICAM-1 dimers (monomers bound together due to antibody labelling) and nano-aggregates binding in resting and CCL21 activated mDCs, normalized to the area quantified and to the background signal outside of the cell. For this, regions of the cell in between the obvious fluorescent ICAM-1 aggregates were selected, the fluorescent intensity was measured using ImageJ, and used to compare the baseline fluorescent signal across all 4 conditions. 20 cells per condition were imaged. ( A–E ) Means ± SEM are depicted. The One-way ANOVA followed by the Tukey multiple comparison test were used to determine significant differences between means. The resulting P values are indicated as follows: ns ( P>0.05); * (P<0.05), ** (P<0.001) and *** (P<0.0001). ( F ) Quantification of bound ICAM-1 nano-aggregates to resting and CCL21 activated mDCs. After applying a threshold of 25% of the fluorescent signal, all visible fluorescent spots per cell were counted. 20 cells per donor and 3 different donors were imaged. Each data point represents the mean value for 1 donor. Means ± SEM and individual data points are depicted, and dotted lines connecting datapoint of the same experiment indicate that not just in average, but in each individual experiment using a different donor, an increase of ICAM-1 nano-aggregate binding is observed after CCL21 activation. The paired two-tailed Student T-test was used to determine significant differences between means. ( G–I ) Representative examples of confocal images of ICAM-1 binding to mDCs: ( G ) dimeric ICAM-1 to resting cells, ( H ) nano-aggregates of ICAM-1 to resting cells and ( I ) nano-aggregates to CCL21 activated cells. Arrows in H and I point to the binding of individual ICAM-1 nano-aggregates to LFA-1.

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Diffusion-based Assay, Control, Binding Assay, Comparison, Activation Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Priming by Chemokines Restricts Lateral Mobility of the Adhesion Receptor LFA-1 and Restores Adhesion to ICAM-1 Nano-Aggregates on Human Mature Dendritic Cells

doi: 10.1371/journal.pone.0099589

Figure Lengend Snippet: ( A, B ) Representative images of ( A ) a monocyte seeded on a TS2/4 pattern, ( B ) a resting mDC on irrelevant IgG1 pattern, ( C ) a resting mDC seeded on a TS2/4 pattern and ( D ) a CCL21 activated mDC seeded on a ICAM-1 pattern. Green corresponds to Talin1 and red to the location of IgG1, TS2/4 or ICAM-1 positive squares. Cells are delineated by white lines. ( E ) Quantification of the degree of Talin1 enhancement to the positive areas in monocytes in different conditions (see Methods). Mean enhancement factor is displayed in red per condition. ( F ) Percentage of positive squares per experiment (n = 3, each a different donor) that showed significantly enhanced Talin1 signal per condition in monocytes. An enhancement factor of ≥1.5 was considered significantly enhanced, since 95% of the control sample (monocytes on IgG1) showed an enhancement factor below this value. ( G ) Quantification of the degree of Talin1 enhancement to the positive areas in mDCs in different conditions (see Methods). Mean enhancement factor is displayed in red per condition. ( H ) Percentage of positive squares per experiment (n = 3, each a different donor) that showed significantly enhanced Talin1 signal per condition in mDCs. Around 60 cells of 3 different donors were analyzed per condition. Monocytes contained 10 positive areas on average per cell, while mDCs contained around 50 positive areas. Means ± SEM are depicted. The Kruskal-Wallis test, followed by Dunn’s multiple comparison test was used to determine significant differences between means in E and G. The One-way ANOVA followed by the Tukey’s multiple comparison test were used to determine significant differences between means in F and H. The resulting P values are indicated as follows: ns ( P>0.05); * (P<0.05) and *** (P<0.0001).

Article Snippet: During the experiments where mDCs were activated, 50 μl CCL21 (final concentration in experiments 1 μg/ml; Recombinant Human CCL21/6Ckine, R&D systems) was added during the measurements and mobility was measured before and up to 10 min after addition at one-minute intervals.

Techniques: Control, Comparison

Journal: Oncology Letters

Article Title: CCR7 regulates ANO6 to promote migration of pancreatic ductal adenocarcinoma cells via the ERK signaling pathway

doi: 10.3892/ol.2018.8962

Figure Lengend Snippet: Effects of CCL21/CCR7 axis and the extracellular signal-regulated kinase inhibitor, U0126, on proliferation and migration. OE cells were pretreated with or without U0126 for 2 h, followed by stimulation with CCL21 for 16 h. (A) Proliferative ability of OE cells and NC cells pretreated with or without CCL21 and U0126. (B) Migratory ability of OE and NC cells pretreated with or without CCL21 and U0126. (C) Transwell migration assay was used to evaluate the migration capacity of different cells (magnification, ×100). **P<0.01. CCL21, C-C motif chemokine ligand 21; CCR7, C-C motif chemokine receptor 7; OD, optical density; CON, untransfected control; NC, blank-vector-transfected negative control; OE, CCR7-overexpressing cells.

Article Snippet: Recombinant human CCL21 was obtained from Cyagen Biosciences, Inc. (Santa Clara, CA, USA).

Techniques: Migration, Transwell Migration Assay, Control, Plasmid Preparation, Transfection, Negative Control

Journal: Oncology Letters

Article Title: CCR7 regulates ANO6 to promote migration of pancreatic ductal adenocarcinoma cells via the ERK signaling pathway

doi: 10.3892/ol.2018.8962

Figure Lengend Snippet: CCR7 regulates ERK expression in different cells. (A) Western blot analysis of pERK in OE and NC cells pretreated with or without CCL21 and U0126. GAPDH was used as a loading control. (B) Transcription levels of ERK1/2 in OE and NC cells with or without pretreatment with CCL21 and U0126. **P<0.01. CCR7, C-C motif chemokine receptor 7; ERK, extracellular signal-regulated kinase; pERK, phosphorylated ERK; CCL21, C-C motif chemokine ligand 21; NC, blank-vector-transfected negative control; OE, CCR7-overexpressing cells.

Article Snippet: Recombinant human CCL21 was obtained from Cyagen Biosciences, Inc. (Santa Clara, CA, USA).

Techniques: Expressing, Western Blot, Control, Plasmid Preparation, Transfection, Negative Control

Journal: Oncology Letters

Article Title: CCR7 regulates ANO6 to promote migration of pancreatic ductal adenocarcinoma cells via the ERK signaling pathway

doi: 10.3892/ol.2018.8962

Figure Lengend Snippet: CCR7 ligand-binding function modulates BxPC-3 cell migration by regulating ANO6 expression. (A) Western blot analysis of ANO6 in OE and NC cells pretreated with or without CCL21 and U0126. GAPDH was used as a loading control. (B) Reverse transcription-quantitative polymerase chain reaction analysis of ANO6 in OE and NC cells with or without pretreatment of CCL21 and U0126. **P<0.01. CCR7, C-C motif chemokine receptor 7; CCL21, C-C motif chemokine ligand 21; ANO6, anoctamin 6; NC, blank-vector-transfected negative control; OE, CCR7-overexpressing cells.

Article Snippet: Recombinant human CCL21 was obtained from Cyagen Biosciences, Inc. (Santa Clara, CA, USA).

Techniques: Ligand Binding Assay, Migration, Expressing, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Plasmid Preparation, Transfection, Negative Control