cpla2  (Cell Signaling Technology Inc)

Journal: Journal of Neuroimmune Pharmacology

Article Title: Formyl Peptide Receptor-2-Suppressed Autophagy Promotes the Migration and Invasion of Human Glioblastoma Cells Through PI3K/Akt Signaling

doi: 10.1007/s11481-026-10284-z

Figure Lengend Snippet: The downregulation of FPR2 leads to G2/M cell cycle arrest in glioma cells and prevents the activation of PI3K/Akt signaling. ( A ) The knockdown of FPR2 expression suppressed the protein expression of cdc2, cyclin B1, and cdc-25C, whereas the expression of cyclin D1 did not obviously change. ( B ) Quantitative images of cdc2, cyclin B1, cdc-25C and cyclin D1 using densitometric analysis with ImageJ software. ( C ) Immunoblot analysis revealed the levels of the PI3K subunits p85 and p110a, as well as the b-catenin, GSK3, and phosphorylated and total protein levels of Akt following the stable knockdown of FPR2. ( D ) Quantification of b-cateninand GSK3 b, phosphorylation and the total levels of Akt and the PI3K subunits p85 and p110a. ( E ) The inhibition of FPR2 does not alter the expression of p-p65NF-kB/p65NF-kB but does increase the expression of cPLA2. ( F ) The levels of p-p65NF-kB/p65NF-kB and cPLA2 were quantified using densitometric analysis with ImageJ software. ( G ) The activity of nuclear NF-kB was measured by ELISA. N=8 per group. ( H ) cPLA2 enzymatic activity was measured as outlined in the Methods section. All the data are presented as the means ± standard deviations and were derived from three separate experiments; * p < 0.05, ** p < 0.01 compared with the si-NC group

Article Snippet: ATG5 (#2630), N-cadherin (#4061), E-cadherin (#14472), vimentin (#5741), Gsk3β (#12456), β-catenin (#8480), CDC2 (#28439), cyclin B1 (#4138), cyclin D1 (#2922),p110α (#4255), p85 (#4292), p-Akt (Ser473) (#4060), Akt (#4685), PARP (#9542), cleavedcaspase-3 (#9661), cleavedcaspase-7 (#9491), cleaved-caspase-8 (#9429), p65NF-κB (#3034), p-p65NF-κB (#3033), cPLA2 (#2832) and horseradish peroxidase-conjugated IgG antibodies (#7074 or #7076) were purchased from Cell Signaling Technology (Danvers, MA, USA).SQSTM1 (#ab207305) and BECN1 (#ab62557) were obtained from Abcam (Cambridge, UK).Cyclin B1 (#AF6000-SP) and Cdc25 (#MAB4459-SP) antibodies were purchased from the American R&D Company (R&D Systems, Minneapolis, MN).

Techniques: Activation Assay, Knockdown, Expressing, Software, Western Blot, Phospho-proteomics, Inhibition, Activity Assay, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Representative immunoblots of dHL60 neutrophils, along with the respective quantification graph showing the level of indicated proteins in the cell lysates. Data are presented as means ± SD and P values calculated using two-way analysis of variance (ANOVA). N = 3. ( B ) Representative airyscan microscopy images of fixed dHL60 neutrophils chemotaxing underagarose showing the nuclear levels of LMNA/C quantified and presented as a scatter plot with mean ± 95% confidence interval (CI) of >30 data points (circles) pooled from three independent experiments. Scale bar, 5 μm; the green line represents the cell boundary. P values determined using ordinary one-way ANOVA are shown. ( C ) Representative airyscan microscopy images of dHL60 neutrophils migrating toward fMLF under agarose, fixed, and immunostained for LBR (magenta), phalloidin (yellow), and Hoechst (blue). Scale bar, 5 μm; the green line represents the cell boundary. ( D and E ) Scatter plots showing the effect of cPLA 2 α on the number of NE folds and nuclei form factor, presented as mean ± 95% CI of >30 data points (circles) pooled from three independent experiments. The top panels show nuclear outlines and LBR folds (D) as segmented using CellProfiler, and nuclear morphology described by form factor (E). P values determined using ordinary one-way ANOVA are shown.

Article Snippet: The electrophoresed proteins were transferred to 0.2-μm polyvinylidene difluoride membrane, blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in tris-buffered saline containing 0.1% Tween-20 (Thermo Fisher Scientific, 337-500), and probed for specific proteins using antibody against FLAP (1 μg/ml, Abcam, 85227), flotillin 2 (1:1,000, CST, 3436), nSMase1 (1:500, CST, 3867), and cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), using electrochemiluminescence capture on photographic films.

Techniques: Western Blot, Microscopy

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Schematic depicting the behavior of neutrophils and their nuclei when plated on sADF fibslips in the presence of fMLF. ( B ) Representative confocal microscopy images of dHL60 neutrophils showing the shape of cells (phalloidin, yellow) and their nuclei (Hoechst, cyan) on aligned fibers (magenta), along the xy and xz axes, 30 min post–fMLF treatment. Scale bar, 5 μm. ( C and D ) Graphs plotted as means ± SEM showing the percent of cells aligned with fibers (C) and the percent of aligned cells with fiber-aligned nuclei (D). N = 3. ( E and F ) Graphs plotted as means ± SEM of data points pooled from three independent experiments, showing the nuclear volume (E) and the height of nuclei (F) within all aligned cells. ( G ) The approach used to calculate the cell and nuclei aspect ratio. The orange outline denotes the cell bounding box, and the black outline denotes the nuclear bounding box. Wavy red lines denote aligned microfibers. ( H and I ) Graphs plotted as means ± SEM of data points pooled from three independent experiments, showing the changes in the nuclear (H) and cellular (I) aspect ratio in Scr, cPLA 2 α KO, and GFP-cPLA 2 α cells. P values determined using repeated measures (RM) one-way ANOVA (C and D) and ordinary one-way ANOVA (E and F and H and I) are shown. ( J ) Graphs showing the correlation of cell and nuclei aspect ratio in dHL60 neutrophils plated on aligned fibers and activated with fMLF for 30 min.

Article Snippet: The electrophoresed proteins were transferred to 0.2-μm polyvinylidene difluoride membrane, blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in tris-buffered saline containing 0.1% Tween-20 (Thermo Fisher Scientific, 337-500), and probed for specific proteins using antibody against FLAP (1 μg/ml, Abcam, 85227), flotillin 2 (1:1,000, CST, 3436), nSMase1 (1:500, CST, 3867), and cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), using electrochemiluminescence capture on photographic films.

Techniques: Confocal Microscopy

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Image of the C 3 showing the cell and chemoattractant inlet/outlet and the migration chamber. The zoomed inset (green) shows the migration chamber, and the zoomed insets orange) show SEM images of the 3- and 5-μm constrictions. ( B ) Image (top) and graph (bottom) showing the diffusion rate and gradient stability of fMLF–Alexa Flour 488 in C 3 . ( C ) Phase contrast images of Scr dHL60 neutrophils migrating toward fMLF through 3-μm constrictions in C 3 at different time points. Scale bar, 100 μm. ( D ) Color-coded tracks of individual cells migrating toward the fMLF, through either 5- or 3-μm constrictions. Refer to the temporal color map on the right. The red dashed line indicates the point of constriction 100 μm from the migration start site. The black dashed line indicates the support site. ( E and F ) Graph showing the percentage of cells entering the C 3 that squeezed through 5- or 3-μm constrictions (E) and the postconstriction Euclidean distance (F). N = 3. ( G ) Graph plotted as means ± SEM showing the change in cell eccentricity in response to constrictions during chemotaxis. N = 3. ( H ) Before-after graph showing the change in speed and directionality of chemotaxing cells post–3-μm constrictions. Randomized data points from three independent experiments are plotted. The P values calculated using multiple paired t test are presented. ( I ) Graphs showing the changes in postconstriction median speed and median directionality of chemotaxing Scr, cPLA 2 α KO, and GFP-cPLA 2 α dHL60 cell populations, compared to preconstriction parameters. N ≥ 3. Graphs are plotted as means ± SEM. The P values determined using three-way ANOVA are shown. ( J ) Before-after graph showing the change in the directionality of chemotaxing GFP-cPLA 2 α expressing cells treated with either DMSO or FLAP inhibitor (MK886) in 3-μm constrictions. P values determined using two-way ANOVA are shown.

Article Snippet: The electrophoresed proteins were transferred to 0.2-μm polyvinylidene difluoride membrane, blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in tris-buffered saline containing 0.1% Tween-20 (Thermo Fisher Scientific, 337-500), and probed for specific proteins using antibody against FLAP (1 μg/ml, Abcam, 85227), flotillin 2 (1:1,000, CST, 3436), nSMase1 (1:500, CST, 3867), and cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), using electrochemiluminescence capture on photographic films.

Techniques: Migration, Diffusion-based Assay, Chemotaxis Assay, Expressing

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Representative microscopy images of dHL60 cells stained with CellMask (PM, magenta) and Hoechst 33342 (nucleus, cyan) 25 min after the start of chemotaxis in 3-μm C 3 devices, showing the morphological transitions from amoeboid-like to keratocyte-like migration mode (see insets). The dashed white line indicates the position of constrictions. Scale bar, 10 μm, and 20 μm in the zoomed insets. N = 2. ( B ) Schematic showing the metrics used for measuring various cellular parameters. ( C ) Graph showing the percentage of keratocytes in Scr versus cPLA 2 α KO cells, and the classifier used to ascertain keratocyte-like morphology.

Article Snippet: The electrophoresed proteins were transferred to 0.2-μm polyvinylidene difluoride membrane, blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in tris-buffered saline containing 0.1% Tween-20 (Thermo Fisher Scientific, 337-500), and probed for specific proteins using antibody against FLAP (1 μg/ml, Abcam, 85227), flotillin 2 (1:1,000, CST, 3436), nSMase1 (1:500, CST, 3867), and cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), using electrochemiluminescence capture on photographic films.

Techniques: Microscopy, Staining, Chemotaxis Assay, Migration

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Representative microscopy images of Fluo4-AM–labeled Scr and cPLA 2 α KO dHL60 neutrophils chemotaxing toward fMLF and traversing either 5- or 3-μm constrictions in C 3 devices. Dashed white line marks the constriction site. Pseudocolor scale (blue-green-yellow) reflects increasing intracellular calcium intensity. Scale bar, 5 μm. ( B ) Single-cell traces of calcium intensity aligned along the direction of chemotaxis. Calcium intensity ( y axis) is plotted against distance traveled ( x axis), with trace color indicating time (yellow, earlier; blue, later). The vertical blue line denotes the constriction position, and gray-blue–shaded regions denote one cell length (25 μm) used to quantify the pre- and postconstriction calcium intensity plotted in (C). ( C ) Scatter plot showing the fold change in postconstriction Fluo4-AM intensity for individual cells, presented as means ± 95% CI. The P values were calculated using ordinary one-way ANOVA; pooled cell numbers from two independent experiments are indicated as n .

Article Snippet: The electrophoresed proteins were transferred to 0.2-μm polyvinylidene difluoride membrane, blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in tris-buffered saline containing 0.1% Tween-20 (Thermo Fisher Scientific, 337-500), and probed for specific proteins using antibody against FLAP (1 μg/ml, Abcam, 85227), flotillin 2 (1:1,000, CST, 3436), nSMase1 (1:500, CST, 3867), and cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), using electrochemiluminescence capture on photographic films.

Techniques: Microscopy, Labeling, Single Cell, Chemotaxis Assay

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Representative confocal microscopy images of dHL60 neutrophils chemotaxing toward fMLF, fixed, and immunostained for pMLC II and Hoechst 33342 (nuclei). Blue outline indicates the cell periphery, and orange outline indicates constriction pillars. Scale bar, 10 μm. ( B ) Graphs plotted as means ± SEM, showing the change in total pMLC II intensity within the cell before, during, and after constrictions. N = 3. ( C ) Representative microscopy image (left) showing the cortex (yellow), nucleus (cyan), and polarized pMLC II (red) outlines. The scatter dot plot (right) shows the cortex-to-cytosol pMLC II intensity ratio in the chemotaxing cells before and after 3-μm constrictions. P values determined using two-way ANOVA are shown. The number of data points ( n ) pooled from three independent experiments are plotted as means ± 95% CI are mentioned on the graph. ( D ) Scatter dot plot showing the cortex-to-cytosol pMLC II intensity ratio in chemotaxing cells before and after 3-μm constrictions, presented as means ± 95% CI. P values determined using ordinary one-way ANOVA are shown, and the number of data points ( n ) pooled from two independent experiments is mentioned. ( E ) Airyscan microscopy images (left) of a GFP-cPLA 2 α dHL60 neutrophil squeezing through a 3-μm constriction, immunostained for cPLA 2 α (magenta) and Hoechst (gray), along with the phase contrast image and individual channels in grayscale. Orange outlines the constriction pillars, and the yellow arrow points to the NE enrichment of cPLA 2 α at the constriction. Scale bar, 3 μm, and 2 μm in the zoomed inset. Scatter dot plot (right) showing the changes in cPLA 2 α levels at the NE before and after 3- and 5-μm constrictions. Data points (circles) from three independent experiments were pooled and plotted as means ± 95% CI, and P values determined using two-way ANOVA are presented.

Article Snippet: The electrophoresed proteins were transferred to 0.2-μm polyvinylidene difluoride membrane, blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in tris-buffered saline containing 0.1% Tween-20 (Thermo Fisher Scientific, 337-500), and probed for specific proteins using antibody against FLAP (1 μg/ml, Abcam, 85227), flotillin 2 (1:1,000, CST, 3436), nSMase1 (1:500, CST, 3867), and cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), using electrochemiluminescence capture on photographic films.

Techniques: Confocal Microscopy, Microscopy

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Fourfold expansion microscopy images of chemotaxing GFP-cPLA 2 α dHL60 cells and immunostained with the indicated antibodies. Orange dashed outlines mark regions used for fluorescence intensity profiling. Histograms show fluorescence intensity profiles averaged across multiple images ( n ) and plotted as means ± SEM. Solid lines represent the mean, and shaded bars indicate the standard error. N = 3. Scale bar, 300 nm. ( B and C ) Three-dimensional volumetric view (B) of fixed and immunostained nuclei isolated from dHL60 neutrophils showing the distribution of GFP-cPLA 2 α (green), ceramide (red), and Hoechst 33342 (blue), quantified and presented as the scatter plot (C) showing Mander’s co-occurrence. Data points ( n ) are plotted as means ± 95% CI. N = 3. Scale bar, 10 μm. ( D and E ) Schematic (D) depicting the steps involved in the isolation of NE-membrane microdomains, and immunoblots (E) showing the cPLA 2 α, FLAP, nSMase1, and Flotillin 2 distribution in DRM and DSM fractions of NE obtained from activated cells. N = 4. ( F and G ) Representative immunoblot (F) and graph (G) plotted as means ± SD, showing the levels of CD63, FLAP, and cPLA 2 α in purified exosomes obtained from various cell lines. N = 3. ( H ) Graph showing the levels of LTB 4 within the exosomes purified from various cell lines upon fMLF activation. N = 5. ( I and J ) Representative immunoblots (I) and graph (J) plotted as means ± SD, showing the levels of cPLA 2 α, 5LO, and Flotillin 2 in the purified exosomes upon trypsin treatment. N = 3. ( K ) Scatter dot plot showing the levels of LTB 4 within exosomes isolated from fMLF-activated PMNs and treated in vitro either with DMSO or MAFP for 30 min at 37°C. Data points (red circles) representing three independent experiments are plotted as means ± SD. The P values determined using Mann-Whitney test (C), two-way ANOVA (G and J), and Student t test (K) are presented.

Article Snippet: The electrophoresed proteins were transferred to 0.2-μm polyvinylidene difluoride membrane, blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in tris-buffered saline containing 0.1% Tween-20 (Thermo Fisher Scientific, 337-500), and probed for specific proteins using antibody against FLAP (1 μg/ml, Abcam, 85227), flotillin 2 (1:1,000, CST, 3436), nSMase1 (1:500, CST, 3867), and cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), using electrochemiluminescence capture on photographic films.

Techniques: Microscopy, Fluorescence, Isolation, Membrane, Western Blot, Purification, Activation Assay, In Vitro, MANN-WHITNEY

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A and B ) Representative airyscan microscopy images of PMNs chemotaxing under agarose, immunostained for cPLA 2 α (magenta) and (A) LBR (yellow) or (B) 5-LO (yellow), and costained with Hoechst 33342 (cyan). Orange outlines indicate cell boundaries. The region within the dashed white rectangle is shown on the right as individual inverted grayscale channels. Fluorescence intensity profiles across the lines masked on zoomed insets (A) near NE bud (bottom line) and away from bud (top line) and (B) on the NE-derived cytosolic vesicle are plotted below the zoomed panels. The line profiles are presented as mean ± SEM of 11 NE-budding sites of 9 cells (A), and 10 MVBs from 7 cells (B). N = 3. Scale bar, 5 μm. ( C and D ) Representative confocal microscopy images (top) of PMNs treated with either DMSO or MAFP (C) and pyrrophenone (D), chemotaxing under agarose, immunostained for pMLC II (magenta), cPLA 2 α (yellow), and costained with Hoechst 33342 (gray). Blue outlines indicate cell boundaries, and the magenta arrow points to polarized pMLC II. Scale bar, 5 μm. Quantification from multiple images is presented as the scatter plot (bottom) showing cortex to cytosol pMLC II intensity and fraction of cPLA 2 α on the NE. Multiple data points ( n ) pooled from two experiments are plotted as a means ± 95% CI, and the P values determined using the Mann-Whitney test are presented.

Article Snippet: The electrophoresed proteins were transferred to 0.2-μm polyvinylidene difluoride membrane, blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in tris-buffered saline containing 0.1% Tween-20 (Thermo Fisher Scientific, 337-500), and probed for specific proteins using antibody against FLAP (1 μg/ml, Abcam, 85227), flotillin 2 (1:1,000, CST, 3436), nSMase1 (1:500, CST, 3867), and cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), using electrochemiluminescence capture on photographic films.

Techniques: Microscopy, Fluorescence, Derivative Assay, Confocal Microscopy, MANN-WHITNEY

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A to C ) Representative confocal images showing differential interference contrast (DIC), pMLC II (heatmap), and LBR (magenta) with Hoechst (gray) in neutrophils chemotaxing through 5- and 3-μm constrictions under the indicated conditions. Yellow and blue arrows denote NE-bud and NE-derived vesicles, respectively. Scatter plots show cortex/cytosol pMLC II ratios versus number of LBR-positive vesicles per cell ( n = number of cells from two independent experiments). The dashed line depicts no change in pMLC II ratio, and the solid black line indicates the slope of the nonlinear regression. The median ± 95% CI of the number of LBR-positive vesicles in each condition for before (blue) and after (red) constrictions is mentioned on the graph, with P value as calculated using uncorrected Dunn’s test. ( D ) Scatter dot plot of cortex/cytosol pMLC II ratio of Scr dHL60 before and after migration through 3- and 5-μm constrictions, presented as means ± 95% CI, with individual data points (circles, n ) pooled from two independent experiments. The P values are calculated using ordinary one-way ANOVA. ( E ) Schematic illustrating the proposed model whereby nuclear squeezing promotes cPLA 2 α recruitment and packaging onto the exofacial surface of NE-derived exosomes, leading to polarized pMLCII activation and persistent neutrophil chemotaxis.

Article Snippet: The electrophoresed proteins were transferred to 0.2-μm polyvinylidene difluoride membrane, blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in tris-buffered saline containing 0.1% Tween-20 (Thermo Fisher Scientific, 337-500), and probed for specific proteins using antibody against FLAP (1 μg/ml, Abcam, 85227), flotillin 2 (1:1,000, CST, 3436), nSMase1 (1:500, CST, 3867), and cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), using electrochemiluminescence capture on photographic films.

Techniques: Derivative Assay, Migration, Activation Assay, Chemotaxis Assay

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Representative immunoblots of dHL60 neutrophils, along with the respective quantification graph showing the level of indicated proteins in the cell lysates. Data are presented as means ± SD and P values calculated using two-way analysis of variance (ANOVA). N = 3. ( B ) Representative airyscan microscopy images of fixed dHL60 neutrophils chemotaxing underagarose showing the nuclear levels of LMNA/C quantified and presented as a scatter plot with mean ± 95% confidence interval (CI) of >30 data points (circles) pooled from three independent experiments. Scale bar, 5 μm; the green line represents the cell boundary. P values determined using ordinary one-way ANOVA are shown. ( C ) Representative airyscan microscopy images of dHL60 neutrophils migrating toward fMLF under agarose, fixed, and immunostained for LBR (magenta), phalloidin (yellow), and Hoechst (blue). Scale bar, 5 μm; the green line represents the cell boundary. ( D and E ) Scatter plots showing the effect of cPLA 2 α on the number of NE folds and nuclei form factor, presented as mean ± 95% CI of >30 data points (circles) pooled from three independent experiments. The top panels show nuclear outlines and LBR folds (D) as segmented using CellProfiler, and nuclear morphology described by form factor (E). P values determined using ordinary one-way ANOVA are shown.

Article Snippet: The proteins were transferred to 0.2-μm nitrocellulose membrane (MDI, SCNX8401XXXX101), blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in 0.1% Tween-20 containing tris-buffered saline, and probed for specific proteins using antibody against cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), FLAP (1 μg ml −1 , Abcam 85227), 5LO (1:1000, Abcam, ab169755), LTA 4 H (1:1000 Protein Tech, 13662-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (1:1000, Santa Cruz Biotechnology).

Techniques: Western Blot, Microscopy

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Schematic depicting the behavior of neutrophils and their nuclei when plated on sADF fibslips in the presence of fMLF. ( B ) Representative confocal microscopy images of dHL60 neutrophils showing the shape of cells (phalloidin, yellow) and their nuclei (Hoechst, cyan) on aligned fibers (magenta), along the xy and xz axes, 30 min post–fMLF treatment. Scale bar, 5 μm. ( C and D ) Graphs plotted as means ± SEM showing the percent of cells aligned with fibers (C) and the percent of aligned cells with fiber-aligned nuclei (D). N = 3. ( E and F ) Graphs plotted as means ± SEM of data points pooled from three independent experiments, showing the nuclear volume (E) and the height of nuclei (F) within all aligned cells. ( G ) The approach used to calculate the cell and nuclei aspect ratio. The orange outline denotes the cell bounding box, and the black outline denotes the nuclear bounding box. Wavy red lines denote aligned microfibers. ( H and I ) Graphs plotted as means ± SEM of data points pooled from three independent experiments, showing the changes in the nuclear (H) and cellular (I) aspect ratio in Scr, cPLA 2 α KO, and GFP-cPLA 2 α cells. P values determined using repeated measures (RM) one-way ANOVA (C and D) and ordinary one-way ANOVA (E and F and H and I) are shown. ( J ) Graphs showing the correlation of cell and nuclei aspect ratio in dHL60 neutrophils plated on aligned fibers and activated with fMLF for 30 min.

Article Snippet: The proteins were transferred to 0.2-μm nitrocellulose membrane (MDI, SCNX8401XXXX101), blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in 0.1% Tween-20 containing tris-buffered saline, and probed for specific proteins using antibody against cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), FLAP (1 μg ml −1 , Abcam 85227), 5LO (1:1000, Abcam, ab169755), LTA 4 H (1:1000 Protein Tech, 13662-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (1:1000, Santa Cruz Biotechnology).

Techniques: Confocal Microscopy

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Image of the C 3 showing the cell and chemoattractant inlet/outlet and the migration chamber. The zoomed inset (green) shows the migration chamber, and the zoomed insets orange) show SEM images of the 3- and 5-μm constrictions. ( B ) Image (top) and graph (bottom) showing the diffusion rate and gradient stability of fMLF–Alexa Flour 488 in C 3 . ( C ) Phase contrast images of Scr dHL60 neutrophils migrating toward fMLF through 3-μm constrictions in C 3 at different time points. Scale bar, 100 μm. ( D ) Color-coded tracks of individual cells migrating toward the fMLF, through either 5- or 3-μm constrictions. Refer to the temporal color map on the right. The red dashed line indicates the point of constriction 100 μm from the migration start site. The black dashed line indicates the support site. ( E and F ) Graph showing the percentage of cells entering the C 3 that squeezed through 5- or 3-μm constrictions (E) and the postconstriction Euclidean distance (F). N = 3. ( G ) Graph plotted as means ± SEM showing the change in cell eccentricity in response to constrictions during chemotaxis. N = 3. ( H ) Before-after graph showing the change in speed and directionality of chemotaxing cells post–3-μm constrictions. Randomized data points from three independent experiments are plotted. The P values calculated using multiple paired t test are presented. ( I ) Graphs showing the changes in postconstriction median speed and median directionality of chemotaxing Scr, cPLA 2 α KO, and GFP-cPLA 2 α dHL60 cell populations, compared to preconstriction parameters. N ≥ 3. Graphs are plotted as means ± SEM. The P values determined using three-way ANOVA are shown. ( J ) Before-after graph showing the change in the directionality of chemotaxing GFP-cPLA 2 α expressing cells treated with either DMSO or FLAP inhibitor (MK886) in 3-μm constrictions. P values determined using two-way ANOVA are shown.

Article Snippet: The proteins were transferred to 0.2-μm nitrocellulose membrane (MDI, SCNX8401XXXX101), blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in 0.1% Tween-20 containing tris-buffered saline, and probed for specific proteins using antibody against cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), FLAP (1 μg ml −1 , Abcam 85227), 5LO (1:1000, Abcam, ab169755), LTA 4 H (1:1000 Protein Tech, 13662-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (1:1000, Santa Cruz Biotechnology).

Techniques: Migration, Diffusion-based Assay, Chemotaxis Assay, Expressing

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Representative microscopy images of dHL60 cells stained with CellMask (PM, magenta) and Hoechst 33342 (nucleus, cyan) 25 min after the start of chemotaxis in 3-μm C 3 devices, showing the morphological transitions from amoeboid-like to keratocyte-like migration mode (see insets). The dashed white line indicates the position of constrictions. Scale bar, 10 μm, and 20 μm in the zoomed insets. N = 2. ( B ) Schematic showing the metrics used for measuring various cellular parameters. ( C ) Graph showing the percentage of keratocytes in Scr versus cPLA 2 α KO cells, and the classifier used to ascertain keratocyte-like morphology.

Article Snippet: The proteins were transferred to 0.2-μm nitrocellulose membrane (MDI, SCNX8401XXXX101), blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in 0.1% Tween-20 containing tris-buffered saline, and probed for specific proteins using antibody against cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), FLAP (1 μg ml −1 , Abcam 85227), 5LO (1:1000, Abcam, ab169755), LTA 4 H (1:1000 Protein Tech, 13662-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (1:1000, Santa Cruz Biotechnology).

Techniques: Microscopy, Staining, Chemotaxis Assay, Migration

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Representative microscopy images of Fluo4-AM–labeled Scr and cPLA 2 α KO dHL60 neutrophils chemotaxing toward fMLF and traversing either 5- or 3-μm constrictions in C 3 devices. Dashed white line marks the constriction site. Pseudocolor scale (blue-green-yellow) reflects increasing intracellular calcium intensity. Scale bar, 5 μm. ( B ) Single-cell traces of calcium intensity aligned along the direction of chemotaxis. Calcium intensity ( y axis) is plotted against distance traveled ( x axis), with trace color indicating time (yellow, earlier; blue, later). The vertical blue line denotes the constriction position, and gray-blue–shaded regions denote one cell length (25 μm) used to quantify the pre- and postconstriction calcium intensity plotted in (C). ( C ) Scatter plot showing the fold change in postconstriction Fluo4-AM intensity for individual cells, presented as means ± 95% CI. The P values were calculated using ordinary one-way ANOVA; pooled cell numbers from two independent experiments are indicated as n .

Article Snippet: The proteins were transferred to 0.2-μm nitrocellulose membrane (MDI, SCNX8401XXXX101), blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in 0.1% Tween-20 containing tris-buffered saline, and probed for specific proteins using antibody against cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), FLAP (1 μg ml −1 , Abcam 85227), 5LO (1:1000, Abcam, ab169755), LTA 4 H (1:1000 Protein Tech, 13662-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (1:1000, Santa Cruz Biotechnology).

Techniques: Microscopy, Labeling, Single Cell, Chemotaxis Assay

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Representative confocal microscopy images of dHL60 neutrophils chemotaxing toward fMLF, fixed, and immunostained for pMLC II and Hoechst 33342 (nuclei). Blue outline indicates the cell periphery, and orange outline indicates constriction pillars. Scale bar, 10 μm. ( B ) Graphs plotted as means ± SEM, showing the change in total pMLC II intensity within the cell before, during, and after constrictions. N = 3. ( C ) Representative microscopy image (left) showing the cortex (yellow), nucleus (cyan), and polarized pMLC II (red) outlines. The scatter dot plot (right) shows the cortex-to-cytosol pMLC II intensity ratio in the chemotaxing cells before and after 3-μm constrictions. P values determined using two-way ANOVA are shown. The number of data points ( n ) pooled from three independent experiments are plotted as means ± 95% CI are mentioned on the graph. ( D ) Scatter dot plot showing the cortex-to-cytosol pMLC II intensity ratio in chemotaxing cells before and after 3-μm constrictions, presented as means ± 95% CI. P values determined using ordinary one-way ANOVA are shown, and the number of data points ( n ) pooled from two independent experiments is mentioned. ( E ) Airyscan microscopy images (left) of a GFP-cPLA 2 α dHL60 neutrophil squeezing through a 3-μm constriction, immunostained for cPLA 2 α (magenta) and Hoechst (gray), along with the phase contrast image and individual channels in grayscale. Orange outlines the constriction pillars, and the yellow arrow points to the NE enrichment of cPLA 2 α at the constriction. Scale bar, 3 μm, and 2 μm in the zoomed inset. Scatter dot plot (right) showing the changes in cPLA 2 α levels at the NE before and after 3- and 5-μm constrictions. Data points (circles) from three independent experiments were pooled and plotted as means ± 95% CI, and P values determined using two-way ANOVA are presented.

Article Snippet: The proteins were transferred to 0.2-μm nitrocellulose membrane (MDI, SCNX8401XXXX101), blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in 0.1% Tween-20 containing tris-buffered saline, and probed for specific proteins using antibody against cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), FLAP (1 μg ml −1 , Abcam 85227), 5LO (1:1000, Abcam, ab169755), LTA 4 H (1:1000 Protein Tech, 13662-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (1:1000, Santa Cruz Biotechnology).

Techniques: Confocal Microscopy, Microscopy

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A ) Fourfold expansion microscopy images of chemotaxing GFP-cPLA 2 α dHL60 cells and immunostained with the indicated antibodies. Orange dashed outlines mark regions used for fluorescence intensity profiling. Histograms show fluorescence intensity profiles averaged across multiple images ( n ) and plotted as means ± SEM. Solid lines represent the mean, and shaded bars indicate the standard error. N = 3. Scale bar, 300 nm. ( B and C ) Three-dimensional volumetric view (B) of fixed and immunostained nuclei isolated from dHL60 neutrophils showing the distribution of GFP-cPLA 2 α (green), ceramide (red), and Hoechst 33342 (blue), quantified and presented as the scatter plot (C) showing Mander’s co-occurrence. Data points ( n ) are plotted as means ± 95% CI. N = 3. Scale bar, 10 μm. ( D and E ) Schematic (D) depicting the steps involved in the isolation of NE-membrane microdomains, and immunoblots (E) showing the cPLA 2 α, FLAP, nSMase1, and Flotillin 2 distribution in DRM and DSM fractions of NE obtained from activated cells. N = 4. ( F and G ) Representative immunoblot (F) and graph (G) plotted as means ± SD, showing the levels of CD63, FLAP, and cPLA 2 α in purified exosomes obtained from various cell lines. N = 3. ( H ) Graph showing the levels of LTB 4 within the exosomes purified from various cell lines upon fMLF activation. N = 5. ( I and J ) Representative immunoblots (I) and graph (J) plotted as means ± SD, showing the levels of cPLA 2 α, 5LO, and Flotillin 2 in the purified exosomes upon trypsin treatment. N = 3. ( K ) Scatter dot plot showing the levels of LTB 4 within exosomes isolated from fMLF-activated PMNs and treated in vitro either with DMSO or MAFP for 30 min at 37°C. Data points (red circles) representing three independent experiments are plotted as means ± SD. The P values determined using Mann-Whitney test (C), two-way ANOVA (G and J), and Student t test (K) are presented.

Article Snippet: The proteins were transferred to 0.2-μm nitrocellulose membrane (MDI, SCNX8401XXXX101), blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in 0.1% Tween-20 containing tris-buffered saline, and probed for specific proteins using antibody against cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), FLAP (1 μg ml −1 , Abcam 85227), 5LO (1:1000, Abcam, ab169755), LTA 4 H (1:1000 Protein Tech, 13662-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (1:1000, Santa Cruz Biotechnology).

Techniques: Microscopy, Fluorescence, Isolation, Membrane, Western Blot, Purification, Activation Assay, In Vitro, MANN-WHITNEY

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A and B ) Representative airyscan microscopy images of PMNs chemotaxing under agarose, immunostained for cPLA 2 α (magenta) and (A) LBR (yellow) or (B) 5-LO (yellow), and costained with Hoechst 33342 (cyan). Orange outlines indicate cell boundaries. The region within the dashed white rectangle is shown on the right as individual inverted grayscale channels. Fluorescence intensity profiles across the lines masked on zoomed insets (A) near NE bud (bottom line) and away from bud (top line) and (B) on the NE-derived cytosolic vesicle are plotted below the zoomed panels. The line profiles are presented as mean ± SEM of 11 NE-budding sites of 9 cells (A), and 10 MVBs from 7 cells (B). N = 3. Scale bar, 5 μm. ( C and D ) Representative confocal microscopy images (top) of PMNs treated with either DMSO or MAFP (C) and pyrrophenone (D), chemotaxing under agarose, immunostained for pMLC II (magenta), cPLA 2 α (yellow), and costained with Hoechst 33342 (gray). Blue outlines indicate cell boundaries, and the magenta arrow points to polarized pMLC II. Scale bar, 5 μm. Quantification from multiple images is presented as the scatter plot (bottom) showing cortex to cytosol pMLC II intensity and fraction of cPLA 2 α on the NE. Multiple data points ( n ) pooled from two experiments are plotted as a means ± 95% CI, and the P values determined using the Mann-Whitney test are presented.

Article Snippet: The proteins were transferred to 0.2-μm nitrocellulose membrane (MDI, SCNX8401XXXX101), blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in 0.1% Tween-20 containing tris-buffered saline, and probed for specific proteins using antibody against cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), FLAP (1 μg ml −1 , Abcam 85227), 5LO (1:1000, Abcam, ab169755), LTA 4 H (1:1000 Protein Tech, 13662-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (1:1000, Santa Cruz Biotechnology).

Techniques: Microscopy, Fluorescence, Derivative Assay, Confocal Microscopy, MANN-WHITNEY

Journal: Science Advances

Article Title: cPLA 2 α targeting to exosomes connects nuclear deformation to LTB 4 -signaling during neutrophil chemotaxis

doi: 10.1126/sciadv.aea2784

Figure Lengend Snippet: ( A to C ) Representative confocal images showing differential interference contrast (DIC), pMLC II (heatmap), and LBR (magenta) with Hoechst (gray) in neutrophils chemotaxing through 5- and 3-μm constrictions under the indicated conditions. Yellow and blue arrows denote NE-bud and NE-derived vesicles, respectively. Scatter plots show cortex/cytosol pMLC II ratios versus number of LBR-positive vesicles per cell ( n = number of cells from two independent experiments). The dashed line depicts no change in pMLC II ratio, and the solid black line indicates the slope of the nonlinear regression. The median ± 95% CI of the number of LBR-positive vesicles in each condition for before (blue) and after (red) constrictions is mentioned on the graph, with P value as calculated using uncorrected Dunn’s test. ( D ) Scatter dot plot of cortex/cytosol pMLC II ratio of Scr dHL60 before and after migration through 3- and 5-μm constrictions, presented as means ± 95% CI, with individual data points (circles, n ) pooled from two independent experiments. The P values are calculated using ordinary one-way ANOVA. ( E ) Schematic illustrating the proposed model whereby nuclear squeezing promotes cPLA 2 α recruitment and packaging onto the exofacial surface of NE-derived exosomes, leading to polarized pMLCII activation and persistent neutrophil chemotaxis.

Article Snippet: The proteins were transferred to 0.2-μm nitrocellulose membrane (MDI, SCNX8401XXXX101), blocked using 1× fish gelatin (Thermo Fisher Scientific, NC0382999) in 0.1% Tween-20 containing tris-buffered saline, and probed for specific proteins using antibody against cPLA 2 α (1:1,000, Santa Cruz Biotechnology, sc-376618), FLAP (1 μg ml −1 , Abcam 85227), 5LO (1:1000, Abcam, ab169755), LTA 4 H (1:1000 Protein Tech, 13662-1-AP), and glyceraldehyde-3-phosphate dehydrogenase (1:1000, Santa Cruz Biotechnology).

Techniques: Derivative Assay, Migration, Activation Assay, Chemotaxis Assay