Journal: Journal of Leukocyte Biology
Article Title: RhoGDI in RBL-2H3 cells acts as a negative regulator of Rho GTPase signaling to inhibit granule exocytosis
doi: 10.1093/jleuko/qiae150
Figure Lengend Snippet: RhoGDI KD increases mast cell degranulation and activated cell morphology. A) Degranulation of RhoGDI KD cell lines was measured by β-hexosaminidase released into supernatants. IgE-sensitized cells were stimulated with DNP-BSA and extracellular β-hexosaminidase was compared to the total of intracellular and extracellular β-hexosaminidase. All values were normalized to the resting state scrambled control. Fold change was calculated as the ratio of stimulated/resting. Knockdown of both RhoGDIs significantly increased degranulation ( P = 0.016; P = 0.0110; P = 0.0006 at 15, 30, and 60 min respectively, comparing RhoGDI1/2 DKD to Sc control; n = 5). B) Analysis of FcεRI surface levels in RhoGDI KD strains. Cells were left resting or stimulated for 30 min, then fixed and stained with an FcεRIα antibody and analyzed by flow cytometry. All values were normalized to the resting state scrambled control. Stimulation slightly decreased surface FcεRI levels for all strains. However, no significant differences were observed in FcεRI surface levels between strains. Flow cytometry was done on four biological replicates for each strain (sample data are provided in , ). C) Cells were imaged by widefield microscopy and stained for nuclei ( blue ), F-actin ( red ), and CD63+ granules ( green ) before ( Resting ) and after stimulation. Bar = 25 µm. In the resting state, Sc control cells are elongated, however RhoGDI KD cells are condensed. When stimulated, all cells from all strains transition to an activated state of spread-out and flattened cells. CD63+ granules were localized to the perinuclear region in all strains in their resting states. Upon stimulation, CD63+ granule staining appeared more punctate throughout the cell body, most clearly seen in the RhoGDI2 KD and RhoGDI1/2 DKD strains. Right panels show quantification of CD63 fluorescence signal in the perinuclear region versus the whole cell. RhoGDI1/2 DKD had increased CD63 signal in the perinuclear region relative to the whole cell compared to the other strains. *** P < 0.001; n ≥ 6 cells. D) Still frames from live cell imaging videos of stimulated RBL-2H3 cells. Cells were imaged by differential interference microscopy. Bar = 20 µm. Sc cells formed large dorsal ruffles; however, RhoGDI1 KD and RhoGDI2 KD formed only small dorsal ruffles ( arrows ). RhoGDI1/2 DKD did not form dorsal ruffles. All cells returned to the resting state after prolonged imaging except RhoGDI1/2 DKD cells which maintained a stimulated state. Videos are available at the following DOIs (shown at 60× speed): Video 1 : 10.6084/m9.figshare.25555983. Video 2 : 10.6084/m9.figshare.25492906. Video 3 : 10.6084/m9.figshare.25492915. Video 4 : 10.6084/m9.figshare.25492927.
Article Snippet: F-actin was labeled with Alexa Fluor 546 phalloidin (Thermo Fisher), RhoGDIs were labeled with anti-RhoGDI1 (Santa Cruz Biotechnology) and anti-RhoGDI2 (Abcam), granules were labeled with mouse monoclonal anti-CD63 antibodies (clone AD1, BioRad) and nuclei labeled with DAPI.
Techniques: Control, Knockdown, Staining, Flow Cytometry, Microscopy, Fluorescence, Live Cell Imaging, Imaging
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