anillin gfp rhoa biosensor  (Addgene inc)

Journal: Nature immunology

Article Title: Dynamic encounters with red blood cells trigger splenic marginal zone B cell retention and function.

doi: 10.1038/s41590-023-01690-z

Figure Lengend Snippet: Fig. 6 | CD97 promotes cell membrane retraction through activation of RhoA. a, HEK293T cells were transfected with CD97 (WT)–GFP, CD97 (T419G)–GFP or CTF–GFP fusion constructs. Longest diameters of individual WT- (n = 62), T419G- (n = 62) and CTF-transfected (n = 63) cells are shown. Each symbol represents one cell. One of three independent experiments with similar results is shown. b, Schematic diagram of an optically trapped ligand-coated bead applying pulling force while in contact with the cell membrane. The cell is also indicated to contain an anillin–GFP RhoA biosensor. c,d, HEK293T cells were cotransduced with anillin–GFP RhoA biosensor and CD97 (WT)–Scarlet, CD97 (T419G)–Scarlet or CD97 (PBM)–Scarlet fusion lentivirus and sorted. c, Cell trap forces of individual WT- (n = 70), T419G- (n = 64) and PBM-transduced (n = 62) cells. d, Normalized active RhoA biosensor fluorescence intensity of WT- (n = 27),

Article Snippet: Anillin–GFP RhoA biosensor was cloned from pEGFP-RhoA Biosensor (a gift from M. Glotzer, University of Chicago, Addgene plasmid 68026) and inserted into the lentiviral vector.

Techniques: Membrane, Activation Assay, Transfection, Construct, Fluorescence

Journal: Nature cancer

Article Title: FoxM1 insufficiency hyperactivates Ect2-RhoA-mDia1 signaling to drive cancer

doi: 10.1038/s43018-020-00116-1

Figure Lengend Snippet: (a) Left: Images of indicated MEFs in metaphase stained with TRITC-Phalloidin. Right: Quantification of mitotic cortical actin intensity of the indicated MEFs (n = 12 independent MEF lines per group). (b) Incidence of slow centrosome movement in the indicated prophases treated Cytochalasin D (Cyto D; actin depolymerizer) or SMIFH2 (pan-Formin inhibitor) for 4 h. Significance is denoted for comparison between the DMSO and treatment groups within each genotype (n = 3 independent MEF lines per group). (c) Left: Images of the indicated MEFs expressing GFP-anillin (RhoA biosensor). Right: Quantification of GFP-anillin intensity at the cell cortex in the indicated MEFs (n = 5 independent MEF lines per group). (d) Quantification of cortical actin intensity in indicated MEFs treated with vehicle (Veh) or 1.5 μg/ml RhoA inhibitor (C3) for 4 h (n = 7 independent MEF lines per group). (e) Incidence of slow prophase centrosome movement and (f) Quantification of cells with non-perpendicular spindles in the indicated MEFs as in d (n = 7 independent MEF lines per group). (g) Western blot analysis of Foxm1−/− MEFs stably transduced with the indicated shRNAs. Western blots are representative of 5 independent MEF lines. (h-l) Foxm1−/− MEFs stably transduced with the indicated shRNAs and then analyzed for cortical actin intensity in metaphase (h), non-perpendicular mitotic spindles (i), slow centrosome movement in prophase, (j), chromosome segregation errors (k), and aneuploid metaphase spreads (l). n = 5 independent MEF lines per group in h-j and 3 independent MEF lines per group in k, l. Data represent mean ± s.e.m. Statistics: a-l one-way ANOVA with Sidak’s correction; Scale bar, 5 μm. See source file for original data and uncropped immunoblots.

Article Snippet: GFP-anillin (RhoA biosensor; Addgene #68026) was sub-cloned into pTSIN PGK-puro2 lentiviral vector.

Techniques: Staining, Expressing, Western Blot, Stable Transfection, Transduction