Journal: Molecular Biology of the Cell

Article Title: Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission

doi: 10.1091/mbc.E12-07-0533

Figure Lengend Snippet: Mitotic distribution of LARG. (A, B) HeLa cells were fixed, and α-tubulin (red), RhoA (red), LARG (green; SC H70), and DNA (blue) were visualized by immunofluorescence microscopy, as described in Materials and Methods . Bar, 11 μm.

Article Snippet: Primary antibody dilutions were anti-LARG, 1:2000 (goat RD AF4737, R&D Systems, Minneapolis, MN; rabbit A301-959A, Bethyl Laboratories, Montgomery, TX; rabbit H70, Santa Cruz Biotechnology, Santa Cruz Biotechnology, CA); rabbit anti-PARP1, 1:2000 (Santa Cruz Biotechnology); mouse anti–α-tubulin, 1:2000 (DM1A; Sigma-Aldrich, St. Louis, MO); and rabbit anti-HSP90 α/β, 1:25,000 (Santa Cruz Biotechnology).

Techniques: Immunofluorescence, Microscopy

Journal: Molecular Biology of the Cell

Article Title: Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission

doi: 10.1091/mbc.E12-07-0533

Figure Lengend Snippet: Depletion of LARG results in apoptosis. ( A) HeLa cells were treated with non- NS or LARG siRNA as described in Materials and Methods . Efficiency of endogenous LARG depletion (>90%) is shown by an immunoblot of cell lysates comparing the LARG signal in LARG siRNA lysates to decreasing equivalents of NS cell lysates. (B) HeLa cells were treated with LARG siRNA and fixed 12 h after release from double-thymidine G1/S synchronization to enrich for cells undergoing mitosis. Cells were stained for α-tubulin (red), cleaved caspase-3 (green), and DNA (blue). Images show examples of normal (top) and apoptotic (bottom) cytokinesis in LARG siRNA–treated cells. Bar, 10 μm. (C) Quantification of the percentage of cleaved caspase-3–positive cells (** p < 0.05, n = 1438 NS si cells, 1021 LARG si cells). (D) HeLa cells were transfected with the indicated siRNA together with expression plasmids for mCherry-LARG (Ch-LARG) or the siRNA-resistant Ch-LARG-5Res and analyzed by immunoblot for PARP-1 cleavage as described in Materials and Methods . The arrows indicate the position of endogenous LARG (endo) and exogenous mCherry-tagged LARG (exo). (E) Quantification of PARP-1 cleavage as fold change over control siRNA–treated cells normalized for HSP90 loading control (* p = 0.0008, ** p < 0.02, n = 3 independent experiments).

Article Snippet: Primary antibody dilutions were anti-LARG, 1:2000 (goat RD AF4737, R&D Systems, Minneapolis, MN; rabbit A301-959A, Bethyl Laboratories, Montgomery, TX; rabbit H70, Santa Cruz Biotechnology, Santa Cruz Biotechnology, CA); rabbit anti-PARP1, 1:2000 (Santa Cruz Biotechnology); mouse anti–α-tubulin, 1:2000 (DM1A; Sigma-Aldrich, St. Louis, MO); and rabbit anti-HSP90 α/β, 1:25,000 (Santa Cruz Biotechnology).

Techniques: Western Blot, Staining, Transfection, Expressing, Control

Journal: Molecular Biology of the Cell

Article Title: Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission

doi: 10.1091/mbc.E12-07-0533

Figure Lengend Snippet: Cells depleted of LARG undergo normal mitotic kinetics but are unable to complete abscission. (A) HeLa cells stably expressing GFP–α-tubulin were treated with the indicated siRNA, synchronized at G1/S by the double-thymidine method, and imaged by live cell microscopy beginning just before initiation of mitosis (times indicated in minutes). Arrow highlights presence of α-tubulin in LARG si cells well after control NS si cells have completed abscission. (B) Quantification of mitotic kinetics as measured by α-tubulin dynamics (* p < 0.0001, n = 97 NS si cells, n = 85 LARG si cells). Total, initiation of mitosis to intercellular α-tubulin resolution; condensation to ingression, initiation of mitosis to completion of contractile ring ingression; ingression to resolution, completion of contractile ring ingression to intercellular α-tubulin resolution. (C) HeLa cells were treated with siRNA, fixed 12 h after release from double thymidine, and stained for α-tubulin (green) and DNA (blue). Arrows highlight intercellular bridges that have completed contractile ring ingression. LARG si inset highlights Flemming body with proper midbody matrix (red arrow) and distal thinning of microtubules in the intercellular bridge (flanking white arrows). Bar, 10 μm. (D) Quantification of cells connected by persistent α-tubulin–containing intercellular bridges as fold over control NS si cells (** p < 0.0005, n ≥1500 cells for each siRNA across three independent experiments).

Article Snippet: Primary antibody dilutions were anti-LARG, 1:2000 (goat RD AF4737, R&D Systems, Minneapolis, MN; rabbit A301-959A, Bethyl Laboratories, Montgomery, TX; rabbit H70, Santa Cruz Biotechnology, Santa Cruz Biotechnology, CA); rabbit anti-PARP1, 1:2000 (Santa Cruz Biotechnology); mouse anti–α-tubulin, 1:2000 (DM1A; Sigma-Aldrich, St. Louis, MO); and rabbit anti-HSP90 α/β, 1:25,000 (Santa Cruz Biotechnology).

Techniques: Stable Transfection, Expressing, Microscopy, Control, Staining

Journal: Molecular Biology of the Cell

Article Title: Leukemia-associated RhoGEF (LARG) is a novel RhoGEF in cytokinesis and required for the proper completion of abscission

doi: 10.1091/mbc.E12-07-0533

Figure Lengend Snippet: Cells depleted of LARG undergo an Aurora-B–dependent delay in abscission kinetics. (A) Asynchronous HeLa cells were treated with siRNA for 72 h. Cells were treated with either DMSO vehicle or 2 μM ZM447439 to inhibit Aurora-B for 2 h before fixing and staining for α-tubulin (green) and DNA (blue). Arrows highlight midbody-stage intercellular bridges. Bar, 10 μm. (B) Quantification of the percentage of midbody-stage intercellular bridges for both NS si and LARG si cells in the presence of vehicle or Aurora-B inhibitor (* p ≤ 0.0012, DMSO vs. ZM447439; DMSO, NS si = 7478 cells, LARG si = 3388 cells; ZM447439, NS si = 7229 cells, LARG si = 4249 cells; across four independent experiments). (C) Percentage of midbody-stage intercellular bridges for LARG si cells normalized by subtraction of NS si cell percentages for each DMSO and ZM447439 treatment (* p = 0.0045).

Article Snippet: Primary antibody dilutions were anti-LARG, 1:2000 (goat RD AF4737, R&D Systems, Minneapolis, MN; rabbit A301-959A, Bethyl Laboratories, Montgomery, TX; rabbit H70, Santa Cruz Biotechnology, Santa Cruz Biotechnology, CA); rabbit anti-PARP1, 1:2000 (Santa Cruz Biotechnology); mouse anti–α-tubulin, 1:2000 (DM1A; Sigma-Aldrich, St. Louis, MO); and rabbit anti-HSP90 α/β, 1:25,000 (Santa Cruz Biotechnology).

Techniques: Staining