Journal: bioRxiv

Article Title: Mesenchymal stromal cells and alpha-1 antitrypsin have a strong synergy in modulating inflammation

doi: 10.1101/2022.11.19.517148

Figure Lengend Snippet: Mesenchymal stromal cell (MSC) isolation and characterization. (a) Illustration of the isolation process. (b) MSCs migrating out from the tissue. (c) MSCs during the expansion phase (Passage 1 to 4). (d) Surface marker expression for P4 MSCs. Negative markers include CD34, CD45, CD11b, CD79A, and HLA-DR. (e) P4 MSCs could be differentiated into adipocytes and osteocytes

Article Snippet: P4 MSCs were characterized with the Human Mesenchymal Stem Cell Verification Flow Kit (R&D Systems), including antibodies for positive markers CD90, CD73, CD105, and negative markers CD45, CD34, CD11b, CD79A, HLA-DR, as well as the Human Mesenchymal Stem Cells Multi-Color Flow Kit (R&D Systems) including antibodies for positive markers CD44, CD106, CD146, and CD166.

Techniques: Isolation, Marker, Expressing

Journal: Frontiers in cell and developmental biology

Article Title: The Wdr1-LIMK-Cofilin Axis Controls B Cell Antigen Receptor-Induced Actin Remodeling and Signaling at the Immune Synapse.

doi: 10.3389/fcell.2021.649433

Figure Lengend Snippet: FIGURE 5 | Wdr1 is important for cSMAC formation and BCR signaling at the immune synapse. A20 D1.3 B cells that had been transfected with control (Ctrl) siRNA or Wdr1 siRNA were added to mHEL-HaloTag-expressing COS-7 APCs. The cells were then fixed at the indicated times and the B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images. Scale bars: 5 µm. (B) For each time point, the percent of cells that had formed a cSMAC, defined as >90% of the total Ag fluorescence intensity being contained in 1-2 clusters, is graphed. Each symbol on the graph represents an independent experiment. Paired t-tests were used to calculate p-values. (C,D) The total fluorescence intensity of the mHEL-HaloTag Ag (C) or pCD79 (D) that was present in clusters at the B cell-APC contact site was quantified for each cell. Each dot is one cell. n >25 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. (E) For each B cell represented in (C,D), the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag. The ratio is graphed. The median (blue line) and interquartile ranges (black box) are shown. The data in (C–E) are from the same experiment, which is representative of four independent experiments. The Mann-Whitney U test was used to calculate p-values for (C–E).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes pCD79 (Cell Signaling Technologies, #5173, 1:200 in PBS + 2% FCS), washed, and then incubated for 30min at room temperature with Alexa Fluor 647-conjugated goat antirabbit IgG secondary antibody (Thermo Fisher, #A21244, 1:400 in PBS + 2% FCS) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher, #A12379, 1:400).

Techniques: Transfection, Control, Expressing, Microscopy, MANN-WHITNEY

Journal: Frontiers in cell and developmental biology

Article Title: The Wdr1-LIMK-Cofilin Axis Controls B Cell Antigen Receptor-Induced Actin Remodeling and Signaling at the Immune Synapse.

doi: 10.3389/fcell.2021.649433

Figure Lengend Snippet: FIGURE 6 | Cofilin is important for cSMAC formation and BCR signaling at the immune synapse. A20 D1.3 B cells that had been transfected with control (Ctrl) siRNA or cofilin siRNA were added to mHEL-HaloTag-expressing COS-7 APCs. The cells were then fixed at the indicated times and the B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images. Scale bars: 5 µm. (B) For each time point, the percent of cells that had formed a cSMAC is graphed. Each symbol represents an independent experiment. Paired t-tests were used to calculate p-values. (C,D) The total fluorescence intensity of the mHEL-HaloTag Ag (C) or pCD79 (D) that was present in clusters at the B cell-APC contact site was quantified for each cell. Each dot is one cell. n >30 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. (E) For each B cell represented in (C,D), the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag. The median (blue line) and interquartile ranges (black box) are shown. The data in (C–E) are from the same experiment, which is representative of three independent experiments. The Mann-Whitney U test was used to calculate p-values for (C–E).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes pCD79 (Cell Signaling Technologies, #5173, 1:200 in PBS + 2% FCS), washed, and then incubated for 30min at room temperature with Alexa Fluor 647-conjugated goat antirabbit IgG secondary antibody (Thermo Fisher, #A21244, 1:400 in PBS + 2% FCS) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher, #A12379, 1:400).

Techniques: Transfection, Control, Expressing, Microscopy, MANN-WHITNEY

Journal: Frontiers in cell and developmental biology

Article Title: The Wdr1-LIMK-Cofilin Axis Controls B Cell Antigen Receptor-Induced Actin Remodeling and Signaling at the Immune Synapse.

doi: 10.3389/fcell.2021.649433

Figure Lengend Snippet: FIGURE 7 | Inhibiting LIMK in A20 D1.3 B cells impairs cSMAC formation and BCR signaling at the immune synapse. A20 D1.3 B cells were pre-treated for 1 h with DMSO or 50 µM LIMKi3 before being added to mHEL-HaloTag-expressing COS-7 APCs. The cells were then fixed at the indicated times and the B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images. Scale bars: 5 µm. (B) For each time point, the percent of cells that had formed a cSMAC is graphed. Each symbol represents an independent experiment. Paired t-tests were used to calculate p-values. (C,D) The total fluorescence intensity of the mHEL-HaloTag Ag (C) or pCD79 (D) that was present in clusters at the B cell-APC contact site was quantified for each cell. Each dot is one cell. n > 67 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. (E) For each B cell represented in (C,D), the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of clustered Ag. The median (blue line) and interquartile ranges (black box) are shown. The data in (C–E) are from the same experiment, which is representative of three independent experiments. The Mann-Whitney U test was used to calculate p-values for (C–E).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes pCD79 (Cell Signaling Technologies, #5173, 1:200 in PBS + 2% FCS), washed, and then incubated for 30min at room temperature with Alexa Fluor 647-conjugated goat antirabbit IgG secondary antibody (Thermo Fisher, #A21244, 1:400 in PBS + 2% FCS) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher, #A12379, 1:400).

Techniques: Expressing, Microscopy, MANN-WHITNEY

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: (A) Schematic representation of a 3-plex SILAC approach for profiling phosphorylation dynamics in resting and BCR-stimulated DG75 cells. DG75 cells were cultured in SILAC medium as indicated and were left untreated, or were BCR-stimulated, for 2, 5, 10, or 20 min. Daudi cells were stimulated for 2 and 10 min. Lysates were mixed in a 1:1:1 ratio and digested with trypsin. Resulting phosphopeptides were enriched by either SCX/TiO2 chromatography (global phosphoproteome analysis; GPome) or phosphotyrosine immunoprecipitation (pY-IP; pYome analysis), and analyzed by LC-MS/MS. For analysis of protein expression levels, proteins were separated by 1D-PAGE, digested with trypsin, and analyzed by LC-MS/MS (see SI Materials and Methods for details). (B) Schematic representation of a 2-plex SILAC approach for profiling phosphorylation changes upon inducible CD79a knockdown or upon SYK inhibition. DG75 cells were cultured in SILAC medium and treated as indicated. Lysates were processed as described in A. (C) DG75 and Daudi cells were loaded with the ratiometric Ca2+-chelator INDO-1-AM and subjected to BCR-induced Ca2+ flux analysis by flow cytometry.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: Cell Culture, Chromatography, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Expressing, Inhibition, Flow Cytometry

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: Tonic BCR signaling. (A) CD79a shRNAs are toxic for BL cell lines. The figure shows the fraction of GFP-positive, shRNA-expressing cells relative to the GFP-negative, shRNA-negative fraction at the times indicated (normalized to day 0). Data are representative of three experiments. (B) CD79a and actin immunoblots of lysates derived from DG75 cells that were treated with doxycycline for 18 h to express either unspecific shRNAs (Control) or shRNAs targeting CD79a. (C) BCR cell surface expression in DG75 control cells or CD79a knockdown cells was monitored by flow cytometry 18 h after shRNA induction. (D and E) Unsupervised clustering analysis of all p-sites that were regulated upon BCR stimulation/CD79a knockdown/SYK inhibition. Values for each p-site (row) in all conditions (columns) are colored based on the z-score of the log2-transformed SILAC ratios.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: shRNA, Expressing, Western Blot, Derivative Assay, Flow Cytometry, Inhibition, Transformation Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Elucidation of tonic and activated B-cell receptor signaling in Burkitt’s lymphoma provides insights into regulation of cell survival

doi: 10.1073/pnas.1601053113

Figure Lengend Snippet: (A) BCR cell surface expression was monitored by flow cytometry (red line, DG75; blue line, Daudi). (B) Scatter plots showing the fold-change of p-sites on mainly serines/threonines (GPome, Left) and mainly tyrosines (pYome, Right) as determined by quantitative MS upon BCR stimulation versus CD79a knockdown and SYK inhibition. Selected phosphorylated proteins and p-sites are highlighted.

Article Snippet: Antibodies against the following proteins were used: SLP65, pSLP65, PLCγ2, pPLCγ2, ERK, pERK, SYK, pSYK, BTK, pBTK, CD79a, pCD79a, CBL, pCBL, ACTN4, actin (all from Cell Signaling Technology), pTyr (4G10; Millipore) and ARFGEF2 (Abcam).

Techniques: Expressing, Flow Cytometry, Inhibition

Journal: iScience

Article Title: Human adult, pediatric and microtia auricular cartilage harbor fibronectin-adhering progenitor cells with regenerative ear reconstruction potential.

doi: 10.1016/j.isci.2022.104979

Figure Lengend Snippet: Figure 2. Expression of putative stem cell markers Using flow cytometry, expression of mesenchymal stromal cell specific markers was measured. Data are represented as mean +/- SEM. Individual data points are also shown. High percentages of cells positive for CD90, CD105, and CD73 were found in adult, pediatric, and microtia populations at passage 4. All populations exhibited a low percentage of expression of a panel of surface markers. This negative marker cocktail consisted of CD11b, CD34, CD45, CD79a, and HLA-DR.

Article Snippet: Antibodies Collagen Type I Abcam ab138492; RRID:AB_2861258 Collagen Type II DSHB II-II6B3; RRID:AB_528165 Goat Anti-Mouse HRP DAKO p0447 Elastin Abcam Ab9519; RRID:AB_2099589 Streptavidin conjugated with HRP DAKO P0397 CD45-PE Mouse IgG1 Clone 2D1 R&D Systems FAB1430P; RRID:AB_2237898 CD34-PE Mouse IgG1 Clone QBEnd10 R&D Systems FAB7227P; RRID:AB_10973177 CD11b-PE Mouse IgG2B Clone 238446 R&D Systems FAB16991P; RRID:AB_416844 CD79A-PE Mouse IgG1 Clone 706931 R&D Systems FAB69201P HLA-DR-PE Mouse IgG1 Clone L203 R&D Systems FAB4869P; RRID:AB_1151931 HRP-conjugated EnVision+ for Rabbit DAKO K4010 CD90-APC R&D Systems FAB7335A CD73-CFS R&D Systems 5795-EN CD105-APC Abcam N/A

Techniques: Expressing, Cytometry, Marker