Journal: Frontiers in Cell and Developmental Biology

Article Title: The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

doi: 10.3389/fcell.2021.647063

Figure Lengend Snippet: Depleting GMFγ reduces cSMAC formation and proximal BCR signaling at the immune synapse. Raji D1.3 B cells were transfected with either control siRNA or GMFγ siRNA and added to COS-7 APCs expressing the mHEL-HaloTag Ag (magenta). The cells were fixed at the indicated times and stained with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79, cyan). The B cell-APC interface was imaged by spinning disk microscopy. (A) Representative images from one of five independent experiments. Scale bars: 5 μm. (B) The total fluorescence intensity of the mHEL-HaloTag Ag that had been gathered into clusters at the B cell-APC contact site was quantified for each B cell and the median values were calculated for each time point. Each symbol on the graph represents the median value for the GMFγ knockdown cells, expressed as a percent of the median value for the control siRNA-transfected cells for the same time point in the same experiment. The differently shaped symbols represent five independent experiments. Paired t -tests were used to calculate p -values. (C) The percent of cells that had formed a cSMAC, defined as > 90% of the total Ag fluorescence intensity being contained in one or two clusters, is graphed. The different symbols represent independent experiments. Paired t -tests were used to calculate p -values. (D) The total fluorescence intensity of pCD79 that was present in clusters at the B cell-APC contact site was quantified for each B cell. The left panel shows representative data from one experiment. Each dot is one cell. n > 31 cells per condition. The median (blue line) and interquartile ranges (black box) are shown. The Mann-Whitney U -test was used to calculate p -values. The right panel shows the results from five independent experiments, presented as in (B) , with n > 30 cells per condition in each experiment. Each symbol represents a single experiment in which the median pCD79 fluorescence intensity for GMFγ-depleted cells is expressed as a percent of the corresponding median value for the control cells. Paired t -tests were used to calculate p -values. (E) For each B cell represented in (D) , the total fluorescence intensity of clustered pCD79 was divided by the total fluorescence intensity of the clustered mHEL-HaloTag Ag. The median (blue line) and interquartile ranges (black box) are shown. The data are presented as in (B , D) . **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p ≤ 0.05; ns, not significant ( p > 0.05).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:200 in PBS + 2% BSA), washed, and then incubated for 30 min at room temperature with PBS + 2% BSA containing Alexa Fluor-647-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, #A21244, 1:400) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific, #A12379, 1:400).

Techniques: Transfection, Control, Expressing, Staining, Microscopy, Fluorescence, Knockdown, MANN-WHITNEY

Journal: Frontiers in Cell and Developmental Biology

Article Title: The Actin-Disassembly Protein Glia Maturation Factor γ Enhances Actin Remodeling and B Cell Antigen Receptor Signaling at the Immune Synapse

doi: 10.3389/fcell.2021.647063

Figure Lengend Snippet: Depleting GMFγ does not impair CD79 phosphorylation induced by soluble anti-Ig antibodies. Cells were transfected with either control siRNA or GMFγ siRNA. (A) Raji D1.3 B cells were stimulated with 20 μg/mL goat anti-mouse IgM to initiate signaling through the D1.3 BCR. (B) Ramos B cells were stimulated with donkey anti-human IgM. Cell lysates were analyzed by immunoblotting with antibodies that recognize the phosphorylated CD79a ITAM (pCD79a), actin (loading control), or GMFγ. The left panels show a representative experiment. The right panels show results from 4 independent experiments. For each sample, the pCD79a band intensity was divided by the corresponding actin band intensity (loading control). The resulting ratios were normalized to that for the 0 min control siRNA cell sample (defined as 1.0) in the same experiment. On the graph, each of the four experiments is indicated by a different symbol. The bars show the mean ± SEM. Paired t -tests were used to calculate p -values. ns, not significant ( p > 0.05).

Article Snippet: The cells were stained for 1 h at room temperature with an antibody that recognizes the phosphorylated CD79 ITAMs (pCD79; Cell Signaling Technologies, #5173, 1:200 in PBS + 2% BSA), washed, and then incubated for 30 min at room temperature with PBS + 2% BSA containing Alexa Fluor-647-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, #A21244, 1:400) plus Alexa Fluor 488-conjugated phalloidin (Thermo Fisher Scientific, #A12379, 1:400).

Techniques: Phospho-proteomics, Transfection, Control, Western Blot