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: Molecular Biology of the Cell

Article Title: Substratum stiffness signals through integrin-linked kinase and β1-integrin to regulate midbody proteins and abscission during EMT

doi: 10.1091/mbc.E21-02-0072

Figure Lengend Snippet: Inducing EMT by exposing cells to TGFβ or ectopically expressing Snail leads to MNC through septin-6. (A) qRT-PCR analysis for Snai1 in NMuMG mouse mammary epithelial cells treated with or without TGFβ. (B) Phase-contrast and fluorescence images and (C) quantification of MNC in cells treated with or without TGFβ. (D) qRT-PCR analysis for Snai1 in cells ectopically expressing GFP or Snail. (E) Phase-contrast and fluorescence images and (F) quantification of MNC in cells ectopically expressing GFP or Snail. (G) qRT-PCR analysis for Sept6 in NMuMG cells ectopically expressing GFP or Snail. (H) qRT-PCR analysis for Sept6 in cells ectopically expressing GFP or septin-6. (I) Phase-contrast and fluorescence images and (J) quantification of MNC in cells ectopically expressing GFP or septin-6. (K) qRT-PCR analysis for Sept6 in cells ectopically expressing shCntl or shSeptin-6. (L) Phase-contrast and fluorescence images and (M) quantification of MNC in cells ectopically expressing shCntl or shSeptin-6 treated with or without TGFβ. Shown are mean ± SD of n = 3–4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 using two-sided Welch’s t test (A, D, G, H, K), two-sided Student’s t test (C, F, J), or two-way Anova with Tukey’s post-hoc test (M). Scale bars, 25 μm.

Article Snippet: NMuMG cells were transfected with the pTK88_GFP-Anillin plasmid (Addgene plasmid # 46354), the scramble-shRNA plasmid (shScr, Addgene plasmid #1864), the shAnillin plasmid (Sigma), or the shβ1 plasmid (Sigma) using FuGENE HD Transfection Reagent (Promega).

Techniques: Expressing, Quantitative RT-PCR, Fluorescence

Journal: Molecular Biology of the Cell

Article Title: Substratum stiffness signals through integrin-linked kinase and β1-integrin to regulate midbody proteins and abscission during EMT

doi: 10.1091/mbc.E21-02-0072

Figure Lengend Snippet: Snail and substratum stiffness increase the expression of midbody proteins. (A) Schematic of the abscission machinery. (B) qRT-PCR analysis for Sept6 in NMuMG mouse mammary epithelial cells cultured on soft or stiff substrata and ectopically expressing GFP or Snail. (C) qRT-PCR analysis for Anln (anillin) and Kif23 (Mklp1) in cells ectopically expressing GFP or Snail. (D) Immunoblotting analysis for anillin and Mklp1 in cells ectopically expressing GFP or Snail. (E) qRT-PCR analysis for Anln and Kif23 in cells cultured on soft or stiff substrata. (F) qRT-PCR analysis for Anln and Kif23 in cells cultured on soft or stiff substrata and ectopically expressing GFP or Snail. Shown are mean ± SD of n = 3-4 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 using two-way ANOVA with Tukey’s post-hoc test (B, F) or two-sided Welch’s t test (C, E).

Article Snippet: NMuMG cells were transfected with the pTK88_GFP-Anillin plasmid (Addgene plasmid # 46354), the scramble-shRNA plasmid (shScr, Addgene plasmid #1864), the shAnillin plasmid (Sigma), or the shβ1 plasmid (Sigma) using FuGENE HD Transfection Reagent (Promega).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Western Blot

Journal: Molecular Biology of the Cell

Article Title: Substratum stiffness signals through integrin-linked kinase and β1-integrin to regulate midbody proteins and abscission during EMT

doi: 10.1091/mbc.E21-02-0072

Figure Lengend Snippet: A soft microenvironment protects against MNC even in cells that ectopically express septin-6 or Mklp1. (A) Phase-contrast and fluorescence images of NMuMG mouse mammary epithelial cells ectopically expressing GFP or septin-6 and cultured on soft or stiff substrata. Red, E-cadherin; white, nuclei. Quantification of MNC in cells ectopically expressing (B) septin-6 or (C) Mklp1 cultured on soft or stiff substrata. Shown are mean ± SD of n = 3 independent experiments. ** P < 0.01, **** P < 0.0001 using two-way ANOVA with Tukey’s post-hoc test. Scale bars, 10 μm.

Article Snippet: NMuMG cells were transfected with the pTK88_GFP-Anillin plasmid (Addgene plasmid # 46354), the scramble-shRNA plasmid (shScr, Addgene plasmid #1864), the shAnillin plasmid (Sigma), or the shβ1 plasmid (Sigma) using FuGENE HD Transfection Reagent (Promega).

Techniques: Fluorescence, Expressing, Cell Culture