human osteoblast sarcoma u2 u2os cell line  (ATCC)

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: UXT is a novel centrosomal component. (A) Colocalization of UXT with the centrosome marker protein γ-tubulin. U2OS cells stably expressing EGFP:UXT (green) were labeled with anti-γ-tubulin (red). Images of cells in the G1, S/G2, prometaphase, metaphase, and anaphase are shown. (B) GFP-UXT is localized to the spindle poles during mitosis. U2OS cells stably expressing EGFP:UXT (green) were stained with an anti-α-tubulin (red) antibody. (C) The FLAG:UXT protein is localized to the centrosome. U2OS cells transfected with FLAG:UXT were immunostained with the mouse anti-FLAG antibody (red) and the rabbit anti-γ-tubulin antibody (green).

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Marker, Stable Transfection, Expressing, Labeling, Staining, Transfection

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: Characterization of the anti-UXT antibodies. (A) The anti-UXT antibody 1B2 can detect both the endogenous UXT and the UXT fusion proteins. Cell lysates from HEK293T cell (lane 1), HEK293T cells transfected with EGFP:UXT (lane 2), and HEK293T cells transfected with FLAG-tagged UXT (lane 3) were separated by 15% SDS-PAGE. The expression of UXT was examined with the anti-UXT antibody 1B2 by Western blot analysis. Note that the anti-UXT antibody 1B2 can detect the endogenous UXT protein (18KD), EGFP:UXT (50KD), and FLAG:UXT (25KD). (B and C) Immunofluorescence detection of the endogenous UXT proteins in U2OS cells using the anti-UXT antibodies. U2OS cells were fixed with methanol and stained with anti-γ-tubulin antibody (green) in combination with either the anti-UXT antibody 15A6 (B, in red) or the anti-UXT antibody 6D3 (C, in red). DNA was stained with DAPI (blue). The arrows indicate the centrosomes.

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Transfection, SDS Page, Expressing, Western Blot, Immunofluorescence, Staining

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: Overexpression of UXT disrupts the centrosome structure. (A) Loss of centrosomal γ-tubulin staining in U2OS cells after overexpression of EGFP:UXT. U2OS cells with transient expression of EGFP:UXT (green) were immunostained with the anti-γ-tubulin antibody (red). DNA was stained with DAPI. Note that the γ-tubulin staining on the centrosome is diminished in cells with over expression of GFP-UXT. (B) Electron microscopic image of disorganized centrosome in U2OS cells with overexpression of EGFP: UXT. U2OS cells, transfected with either EGFP or GFP-UXT, were processed for electron microscope imaging. Top, image of the centrosome in U2OS cells transfected with EGFP (control). Bottom, image of the abnormal centrosome in U2OS cells transfected with EGFP:UXT. The arrow indicates a normal centriole.

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Over Expression, Staining, Expressing, Transfection, Microscopy, Imaging, Control

Journal:

Article Title: UXT Is a Novel Centrosomal Protein Essential for Cell Viability D⃞

doi: 10.1091/mbc.E05-08-0705

Figure Lengend Snippet: UXT siRNA knockdown causes cell death. Two nonoverlapping UXT siRNAs were tested and produced similar results. The data obtained with one UXT siRNA were shown. The data obtained with another UXT siRNA were shown as supplemental data (Figure S2). (A) Efficacy of siRNA knockdown of the endogenous UXT protein. U2OS cells were treated with transfection reagent (NT), nonspecific siRNA (NS siRNA), or siRNA for UXT (UXT siRNA), respectively. The cell lysates were subject to Western blot using either the anti-UXT antibody 1B2 (top) or the anti-β-tubulin antibody (bottom). The protein levels of UXT were specifically reduced after 72-h treatment with siRNA for UXT. (B) UXT knockdown inhibits cell proliferation. U2OS cells were treated with nonspecific siRNA (left) or siRNA for UXT (right). Pictures were taken 72 h later. (C) UXT knockdown leads to cell death. U2OS cells were treated with nonspecific RNA or the UXT siRNA oligos. Seventy-two hours later, all the cells were collected and used for propidium iodide staining of DNA content by fluorescence-activated cell sorting. (D) p53 is not required for cell death caused by UXT knockdown. The HCT116 (p53+/+) or p53-negative HCT116 (p53-/-) cells were treated with either the control siRNA or the UXT siRNA. Seventy-two hours after transfection, the percentage of cell death was assessed by the trypan blue assay. The diagram shows the representative results of three experiments.

Article Snippet: Cell Culture and Treatments The human osteoblast sarcoma U2 (U2OS) cell line and the human embryonic kidney 293T (HEK293T) cell line were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Knockdown, Produced, Transfection, Western Blot, Staining, Fluorescence, FACS, Control