Journal: PLoS ONE

Article Title: The Inhibition of Mast Cell Activation of Radix Paeoniae alba Extraction Identified by TCRP Based and Conventional Cell Function Assay Systems

doi: 10.1371/journal.pone.0155930

Figure Lengend Snippet: (A-B) IgE-mediated TCRP comparison with cell-electrode impedance dynamics induced by adenosine or PMA. RBL-2H3 cells were seeded on the E-plates overnight at a density of 20,000 cells/well, and were then treated with DNP-BSA (IgE-sensitized) and adenosine or PMA (no sensitization). (C) Representative distribution of CD63 stimulated by different antigens in RBL-2H3. RBL-2H3 cells with or without IgE sensitization were stimulated with DNP-BSA, adenosine, and PMA for 30 min. Bar, 5 μm. (D) Adenosine induced microtubule formation whereas PMA induced F-actin disassembly. Treated cells were fixed and processed for staining with phalloidin-rhodamine (red) and antibody to β-tubulin (green). Bar, 5 μm. (E) Adenosine together with PMA induced β-hexosaminidase release. The cells were seeded in 24-well plates overnight, pretreated with PMA or adenosine and subsequently activated with different concentrations of adenosine or PMA. *** P < 0.001 vs. with PMA or adenosine only.

Article Snippet: The cells were permeabilized in phosphate-buffered saline (PBS) containing 0.5% Triton X-100 for 15 min and blocked with PBS containing 1% BSA for 1 h. The following primary and secondary stainings were performed: CD63 antibody (Epitomics, Inc.; Burlingame, CA) at a dilution of 1:50, fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG (MultiSciences Biotech Co.; Hangzhou, China) at 1:100, FITC-conjugated tubulin antibody (Cell Signaling Technology; Beverly, MA), phalloidin-rhodamine (Invitrogen Molecular Probes; Carlsbad, CA), and 4′6-diamidino-2-phenylindole (DAPI; Invitrogen).

Techniques: Staining

Journal: Nature Communications

Article Title: Actin nucleation at the centrosome controls lymphocyte polarity

doi: 10.1038/ncomms10969

Figure Lengend Snippet: ( a ) Left: representative images of non-polarized (BCR-ligand − ) and polarized (BCR-ligand + ) B cells. B cells were incubated for 60 min with beads coated with either BCR ligands or with proteins that do not engage the BCR, fixed and the centrosome was stained (γ-tubulin). White circles indicate bead position. Scale bar, 3 μm. Middle: schematics depicting centrosome polarity index measurement. Right: quantification of centrosome polarity index. Data are pooled from three independent experiments with n =80 and 85 cells for BCR-ligand − and BCR-ligand + , respectively. Unpaired Student's t -test was used to determine statistical significance. ( b ) SILAC-based MS workflow used to identify proteins differentially associated with the centrosome of B cells stimulated with either BCR-ligand − or BCR-ligand + beads. ( c ) Western blots highlighting centrosome-containing fractions after centrosome isolation on discontinuous sucrose gradient. Immunoblots are representative of three independent experiments. ( d ) Volcano plot showing the 835 proteins considered for further analysis (light red) among the total of the 1,600 quantified proteins. Horizontal red line represents the threshold for statistical significance (adjusted P value ≤0.05). Vertical red lines represent the biological threshold used to select proteins (−10% and +10% of protein fold change). ( e ) Protein fold change (%) for each of the 45 proteins belonging to the ‘centrosome/microtubule' subgroup (top) and the 43 belonging to the ‘actin' one (bottom).

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit anti-γ-tubulin (Abcam, #Ab11317, 1/2000); fluorescein isothiocyanate (FITC)-conjugated mouse anti-α-tubulin (Abcam, #Ab64503, 1/100); rabbit anti-Arp2 (Abcam, #Ab47654, 1/200); rabbit anti-HS1 and rabbit anti-phospho-HS1 (both from Cell Signalling Technologies, #4557S and #8714P, respectively, 1/50); rabbit anti-WASH (home-made as previously described , 1/250); and human anti-green fluorescent protein (GFP) and anti-red fluorescent protein (RFP) (Recombinant Antibodies Platform, Institut Curie, Paris, France, 1/200).

Techniques: Incubation, Staining, Western Blot, Isolation

Journal: Nature Communications

Article Title: Actin nucleation at the centrosome controls lymphocyte polarity

doi: 10.1038/ncomms10969

Figure Lengend Snippet: ( a , d ) Representative images of B cells under resting conditions or stimulated with BCR-ligand + beads for indicated time, fixed and co-stained for Arp2 (white arrow: cortical pool; *: centrosomal pool) ( a ) or F-actin (phalloidin) ( d ) and the centrosome (α-tubulin). White circles indicate bead position. Dashed grey squares indicate the centrosomal region magnified below each image. Dashed circles on bottom panel highlight the centrosomal area used for quantification. Scale bars, top: 3 μm; bottom: 1 μm. ( b ) Schematics depicting the pipeline used to quantify centrosome-associated Arp2 and F-actin. ( c , e ) Quantification of centrosome-associated Arp2 ( c ) and F-actin ( e ) from cells shown in a and d , respectively. Data are pooled from three independent experiments with ( c ) n =67, 62, 64, 72, 61 and 69 cells, and ( e ) n =54, 63, 64, 62, 59 and 64 cells from left to right, respectively. fluo. int., fluorescence intensity. cent, centrosome. Rest., Resting.

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit anti-γ-tubulin (Abcam, #Ab11317, 1/2000); fluorescein isothiocyanate (FITC)-conjugated mouse anti-α-tubulin (Abcam, #Ab64503, 1/100); rabbit anti-Arp2 (Abcam, #Ab47654, 1/200); rabbit anti-HS1 and rabbit anti-phospho-HS1 (both from Cell Signalling Technologies, #4557S and #8714P, respectively, 1/50); rabbit anti-WASH (home-made as previously described , 1/250); and human anti-green fluorescent protein (GFP) and anti-red fluorescent protein (RFP) (Recombinant Antibodies Platform, Institut Curie, Paris, France, 1/200).

Techniques: Staining, Fluorescence

Journal: Nature Communications

Article Title: Actin nucleation at the centrosome controls lymphocyte polarity

doi: 10.1038/ncomms10969

Figure Lengend Snippet: ( a ) Representative images of actin asters nucleated from isolated centrosomes (white arrow). Scale bar, 8 μm. ( b ) Actin nucleation efficiency was calculated as the ratio of the number of actin asters divided by the number of γ-tubulin spots ( > 200 actin asters and > 450 γ-tubulin spots per condition pooled from four independent experiments). ( c ) Sequential images of F-actin assembly by centrosomes isolated from B cells stimulated with indicated beads. Scale bar, 5 μm. ( d ) Quantification of F-actin nucleation activity ( n= 14 and 12 actin asters per condition; data are representative of four independent experiments) ( e ) Quantification of Arp2 fluorescence (fluo.) intensity associated with purified centrosomes ( n =100 and 190 centrosomes per condition pooled from two independent experiments). ( f ) Quantification of actin nucleation activity of centrosomes purified from resting lymphocytes in the presence of CK666 or dimethylsulfoxide (DMSO; n =12 and 22 actin asters, respectively; data are representative of two independent experiments). ( g ) Representative images of resting B cells treated with DMSO or CK666 for 60 min, fixed and co-stained for F-actin (phalloidin) and the centrosome (α-tubulin). Dashed grey squares indicate the region magnified on the right panel. Dashed circles on the right panel highlight the centrosomal area used for quantification. Scale bars, left: 3 μm; right: 1 μm. ( h ) Quantification of centrosome-associated F-actin of B cells pretreated for 30 min with DMSO or CK666 before being stimulated with indicated beads for 60 min in presence of the drug (>60 cells per condition pooled from three independent experiments). ( i ) Left: sequential images of resting B cells co-transfected with the centrosomal marker eGFP–Centrin1 and the F-actin probe Utrophin-RFP imaged by time-lapse spinning disk microscopy and treated with either DMSO or CK666 (between t −5 min and t 0 min ). Scale bar, 3 μm. Right: quantification of centrosome-associated F-actin over time. Centrosomal F-actin mean fluorescence intensity (MFI) was normalized with respect to initial intensity ( t −10 min ) for each cell. Data are pooled from two independent experiments and graph shows the mean±s.e.m. of n =8 and 10 cells for DMSO and CK666, respectively. P values determined by Mann–Whitney test.

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit anti-γ-tubulin (Abcam, #Ab11317, 1/2000); fluorescein isothiocyanate (FITC)-conjugated mouse anti-α-tubulin (Abcam, #Ab64503, 1/100); rabbit anti-Arp2 (Abcam, #Ab47654, 1/200); rabbit anti-HS1 and rabbit anti-phospho-HS1 (both from Cell Signalling Technologies, #4557S and #8714P, respectively, 1/50); rabbit anti-WASH (home-made as previously described , 1/250); and human anti-green fluorescent protein (GFP) and anti-red fluorescent protein (RFP) (Recombinant Antibodies Platform, Institut Curie, Paris, France, 1/200).

Techniques: Isolation, Activity Assay, Fluorescence, Purification, Staining, Transfection, Marker, Microscopy, MANN-WHITNEY

Journal: Nature Communications

Article Title: Actin nucleation at the centrosome controls lymphocyte polarity

doi: 10.1038/ncomms10969

Figure Lengend Snippet: ( a ) Western blot showing the phosphorylation of HS1 during the course of B-cell stimulation. Representative of two independent experiments. ( b ) Representative images of B cells stimulated with indicated beads for 15 min, fixed and co-stained for total HS1 (top) or phosphorylated HS1 (bottom) and the centrosome (α-tubulin). Dashed white circles indicate bead position. Scale bar, 3 μm. Images are representative of two independent experiments. ( c ) Representative images of B cells stimulated with indicated beads for 60 min, fixed and co-stained for Arp2 and the centrosome (α-tubulin). Dashed white circles indicate bead position. Scale bar, 3 μm. Representative of three independent experiments. ( d , e ) Quantification of synapse-associated Arp2 ( d ) and F-actin ( e ). ( d ) n =71, 64, 68, 72 and 69 cells and ( e ) n =55, 60, 66, 59 and 57 cells from left to right, pooled from three independent experiments. ( f ) Left: sequential images of B cells co-transfected with the centrosomal marker eGFP–Centrin1 and the F-actin probe Utrophin-RFP stimulated with BCR-ligand + beads and imaged by time-lapse spinning disk microscopy. White arrows indicate F-actin clearance from the centrosome (bottom) and its concomitant accumulation at the immune synapse (top) during the course of B-cell stimulation. Dashed white circles indicate bead position. Scale bar, 3 μm. Right: quantification of synapse- (blue) and centrosome- (red) associated F-actin of B cells stimulated with indicated beads. F-actin mean fluorescence intensity (MFI) was normalized with respect to initial intensity ( t −5 min ) for each cell. Data are pooled from two independent experiments and graph shows the mean±s.e.m. of n =7 cells per condition. ( g , h ) Quantification of Arp2 ( g ) and F-actin ( h ) associated with the synapse (left) and the centrosome (right) in control and HS1-silenced B cells stimulated for 60 min with indicated beads. Data are pooled from two ( g ) and three ( h ) independent experiments with ( g ) synapse: n =51, 46 and 53 cells; centrosome: n =51, 52 and 47 cells; and ( h ) synapse: n =72, 74 and 66 cells; centrosome: n =73, 72 and 67 cells from left to right. P values determined by Mann–Whitney test. fluo. int., fluorescence intensity. syn, synapse.

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit anti-γ-tubulin (Abcam, #Ab11317, 1/2000); fluorescein isothiocyanate (FITC)-conjugated mouse anti-α-tubulin (Abcam, #Ab64503, 1/100); rabbit anti-Arp2 (Abcam, #Ab47654, 1/200); rabbit anti-HS1 and rabbit anti-phospho-HS1 (both from Cell Signalling Technologies, #4557S and #8714P, respectively, 1/50); rabbit anti-WASH (home-made as previously described , 1/250); and human anti-green fluorescent protein (GFP) and anti-red fluorescent protein (RFP) (Recombinant Antibodies Platform, Institut Curie, Paris, France, 1/200).

Techniques: Western Blot, Cell Stimulation, Staining, Transfection, Marker, Microscopy, Fluorescence, MANN-WHITNEY

Journal: Nature Communications

Article Title: Actin nucleation at the centrosome controls lymphocyte polarity

doi: 10.1038/ncomms10969

Figure Lengend Snippet: ( a – c ) Quantification of centrosome polarity index of control and HS1-silenced ( a ), CK666-treated ( b ) or HS1-silenced and CK666-treated ( c ) B cells stimulated with indicated beads for 60 min. ( a ) n =77, 71 and 75 cells; ( b ) n =103, 80 and 87 cells and ( c ) n =77, 69, 72 and 75 cells from left to right, pooled from three independent experiments. ( d ) Representative images of control and HS1-silenced B cells treated with dimethylsulfoxide (DMSO) or CK666, stimulated with BCR-ligand + beads for 60 min, fixed and co-stained for F-actin (phalloidin) and the centrosome (α-tubulin). Scale bars, left: 3 μm; right: 0.9 μm. ( e ) Quantification of centrosome-associated F-actin from cells shown in d ( n =41, 47, 38 and 45 cells from left to right, pooled from two independent experiments). ( f ) Quantification of centrosomal F-actin of control, HS1- and HS1 plus Arp2-silenced B cells stimulated for 60 min with indicated beads ( n =58, 65, 65 and 63 cells from left to right, pooled from three independent experiments). ( g ) Correlation analysis of centrosome-associated F-actin and the bead–centrosome distance ( n =185 cells). Spearman correlation test, P <0,0001. ( h ) Left: schematics depicting the construct used to over-activate the Arp2/3 complex at the centrosome (bottom). Right: representative images of control and eGFP–Centrin1–VCA-expressing B cells, stimulated with indicated beads for 60 min, fixed and co-stained for F-actin (phalloidin) and the centrosome (GFP). Scale bar, 3 μm. ( d , h ) White circles indicate bead position. Dashed grey squares indicate the region magnified on the right. Dashed circles on magnifications highlight the centrosomal area used for quantification. ( i , j ) Quantification of centrosomal F-actin ( i ) and centrosome polarity index ( j ) of cells shown in h . ( i ) n =74, 66, 68 and 64 cells and ( j ) n =75, 71 and 64 cells from left to right, pooled from three independent experiments. ( k ) Quantification of centrosome polarity index of control and eGFP–Centrin1–VCA-expressing B cells, treated or not with CK666 and stimulated with indicated beads for 60 min ( n =41, 39, 42 and 42 cells from left to right, pooled from two independent experiments). P values determined by Mann–Whitney test. Cent-VCA, eGFP-centrin1-VCA.

Article Snippet: The following primary antibodies were used for immunofluorescence: rabbit anti-γ-tubulin (Abcam, #Ab11317, 1/2000); fluorescein isothiocyanate (FITC)-conjugated mouse anti-α-tubulin (Abcam, #Ab64503, 1/100); rabbit anti-Arp2 (Abcam, #Ab47654, 1/200); rabbit anti-HS1 and rabbit anti-phospho-HS1 (both from Cell Signalling Technologies, #4557S and #8714P, respectively, 1/50); rabbit anti-WASH (home-made as previously described , 1/250); and human anti-green fluorescent protein (GFP) and anti-red fluorescent protein (RFP) (Recombinant Antibodies Platform, Institut Curie, Paris, France, 1/200).

Techniques: Staining, Construct, Expressing, MANN-WHITNEY