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