Journal: Nucleus (Austin, Tex.)

Article Title: Coilin is rapidly recruited to UVA-induced DNA lesions and γ-radiation affects localized movement of Cajal bodies.

doi: 10.4161/nucl.29229

Figure Lengend Snippet: Figure 1. Nuclear pattern of Cajal bodies in several cell types. (A) Cajal body (green) morphology was studied in mouse embryonic fibroblasts (MEFs), multiple myeloma cells (OPM2 and MOLP8), K562 leukemia cells, and U2OS human osteosarcoma cells. (B) Nuclear distribution of coilin in embryonic stem cells. Coilin was homogeneously distributed in interphase nuclei of human ESCs, or accumulated into body-like structures (Cajal bodies) in hESCs. (C) Cajal bodies were observed in pluripotent mouse ESCs (line D3), and in GOWT1 mESCs and in their differentiated counterparts. (D) Morphological association between Cajal bodies (green) and γ-H2AX-positive foci (red) in non-irradiated and γ-irradiated U2OS cells.

Article Snippet: We analyzed the nuclear pattern of Cajal bodies in mouse embryonic stem cells (mESCs) (line D3, ATCC), in GOWT1 cells (gift from Dr Hitoshi Niwa), and in human ESCs (hESCs).31 Differentiation of mouse ESCs was induced by alltrans retinoic acid according to previously published protocols.32 We also used HeLa cells stably expressing GFP-colin (a generous gift from the laboratory of Prof Angus Lamond, University of Dundee16).

Techniques: Irradiation

Journal: Nucleus (Austin, Tex.)

Article Title: Coilin is rapidly recruited to UVA-induced DNA lesions and γ-radiation affects localized movement of Cajal bodies.

doi: 10.4161/nucl.29229

Figure Lengend Snippet: Figure 5. Recruitment of coilin to UVA-induced DNA lesions. (A) In live U2OS cells transiently expressing GFP-coilin and HeLa cells stably expressing GFP-coilin. Coilin was recruited to micro-irradiated genomic regions. This was also observed in transiently transfected D3 mESCs. These experiments were performed with BrdU pre-sensitization. (B) Recruitment of coilin to UVA-induced DNA lesions in HeLa cells stably expressing GFP-coilin, but lacking CBs. (C) Cells were locally irradiated using a UVA-laser in defined regions of interest (ROI) and endogenous coilin was analyzed under conditions of (a) BrdU pre-sensitization or (b) without BrdU pre-sensitization. (c) CPDs were analyzed without BrdU pre-sensitization. CPDs were visualized by red fluorescence and CBs as green. Experiments were performed in HeLa cells stably expressing GFP-coilin (green). The cell nuclei were clearly visible before local micro- irradiation, and GFP fluorescence was maintained after formaldehyde fixation and immunostaining. To assess endogenous coilin levels, HeLa cells were micro-irradiated and endogenous coilin and CPDs were visual- ized using antibodies (panel Cc).

Article Snippet: We analyzed the nuclear pattern of Cajal bodies in mouse embryonic stem cells (mESCs) (line D3, ATCC), in GOWT1 cells (gift from Dr Hitoshi Niwa), and in human ESCs (hESCs).31 Differentiation of mouse ESCs was induced by alltrans retinoic acid according to previously published protocols.32 We also used HeLa cells stably expressing GFP-colin (a generous gift from the laboratory of Prof Angus Lamond, University of Dundee16).

Techniques: Expressing, Stable Transfection, Irradiation, Transfection, Fluorescence, Immunostaining

Journal: Nucleus (Austin, Tex.)

Article Title: Coilin is rapidly recruited to UVA-induced DNA lesions and γ-radiation affects localized movement of Cajal bodies.

doi: 10.4161/nucl.29229

Figure Lengend Snippet: Figure 9. Localized movement of Cajal bodies. (A) Average enclosing ellipses (yellow) calculated for Cajal bodies in HeLa cells, D3 mESCs, and U2OS cells. Non-treated cells were used as control cell population, and data were compared with average enclosing ellipse of Cajal bodies in cells irradiated by 5 Gy of γ-rays. Tracking of ellipse centroids is shown in blue. Selected ellipses are enlarged in individual frames. Small red dots represent the starting points of tracking analysis. (B) Data represent average enclosing ellipse areas in non-irradiated and γ-irradiated HeLa cells, D3 mESCs, and U2OS cells. (C) Average enclosing ellipse area per Cajal body area in HeLa cells, D3 mESCs, and U2OS cells. Data in panels B and C represent mean ± standard error (S.E.). Asterisks in panels (B) and (C) show statistically significant differences from control values at P ≤ 0.05. Analysis was performed by Student’s t test.

Article Snippet: We analyzed the nuclear pattern of Cajal bodies in mouse embryonic stem cells (mESCs) (line D3, ATCC), in GOWT1 cells (gift from Dr Hitoshi Niwa), and in human ESCs (hESCs).31 Differentiation of mouse ESCs was induced by alltrans retinoic acid according to previously published protocols.32 We also used HeLa cells stably expressing GFP-colin (a generous gift from the laboratory of Prof Angus Lamond, University of Dundee16).

Techniques: Control, Irradiation

Journal: Nucleus (Austin, Tex.)

Article Title: Coilin is rapidly recruited to UVA-induced DNA lesions and γ-radiation affects localized movement of Cajal bodies.

doi: 10.4161/nucl.29229

Figure Lengend Snippet: Figure 8. FRAP analysis of coilin diffusion in various nuclear regions. Kinetics of GFP-coilin were studied in nucleoli, DNA lesions, nucleoplasm, and non-irradiated Cajal bodies in (A) D3 mESCs; (B) U2OS cells; and (C) HeLa cells. FRAP data were normalized to 1, and data represent means ± standard error (S.E.).

Article Snippet: We analyzed the nuclear pattern of Cajal bodies in mouse embryonic stem cells (mESCs) (line D3, ATCC), in GOWT1 cells (gift from Dr Hitoshi Niwa), and in human ESCs (hESCs).31 Differentiation of mouse ESCs was induced by alltrans retinoic acid according to previously published protocols.32 We also used HeLa cells stably expressing GFP-colin (a generous gift from the laboratory of Prof Angus Lamond, University of Dundee16).

Techniques: Diffusion-based Assay, Irradiation

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: ( a ) Representative phase images are shown of WT ESCs and VIM −/− ESCs cultured on MEF feeder layers. Arrows indicate refractive edges of cell colonies. Scale bar represents 200 μm. ( b ) Gene expression of Nanog, Oct4 , and Sox2 ( all normalized to Gapdh ) are shown for both cell types. Data are presented as mean ± SEM (n = 3). ( c ) Flow cytometry analysis for both cell types of NANOG, OCT3/4, and SOX2 are shown. Shaded histograms are for staining (secondary antibody-only) controls. Values listed are for the percentage of cells within the population considered to be positive. WT ESCs are represented in black and VIM −/− ESCs are in red.

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Cell Culture, Expressing, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: ( a ) Phase images for WT EBs and VIM −/− EBs at Day 1 before (left image) and after removal (right image) from the microwells, as well as at Day 2, 4, 6 in suspension culture. All images are at the same magnification and the scale bar represents 400 μm. ( b ) Cross sectional areas for WT EBs and VIM −/− EBs were calculated from phase images (n = 50 EBs per group). ( c ) Immunohistochemical analysis indicates the proliferation marker Ki67 (green) with a nuclear counterstain (blue). Scale bar represents 200 μm.

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Immunohistochemical staining, Marker

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: ( a ) Histological sections were stained for ECAD protein (green) with a nuclear counterstain (blue). The star indicates staining within the interior of a WT EB and arrows indicate discontinuous ECAD expression along VIM −/− EB outer layers. All images are at the same magnification and the scale bar represents 200 μm. ( b ) SEM images were taken of both whole and fractured WT EBs and VIM −/− EBs. Length of scale bars is indicated in each image.

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Staining, Expressing

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: Gene expression of Nanog, Oct4 , and Sox2 (all normalized to Gapdh ) are shown for WT EBs and VIM −/− EBs over 7 days of differentiation as EBs. Data presented as mean ± SEM (n = 3).

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Expressing

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: ( a ) Gene expression of Brachy-T (mesodermal commitment), as well as Meox1 (paraxial mesoderm) and Flk1 (lateral plate mesoderm) are shown for WT EBs and VIM −/− EBs over 7 days of differentiation (all normalized to Gapdh ). ( b ) Immunohistochemical analysis of FLK1 protein expression (green) with a nuclear counterstain (blue) in EBs at Days 6, 8, and 10. All images were taken at the same magnification and the scale bar represents 200 μm. ( c ) A representative histogram of FLK1 protein expression is shown for WT EBs (black) and VIM −/− EBs (red), as well as their respective secondary antibody-only controls (shaded histograms). The bar graph shows the percentage of positive cells for each group at Day 10. Data are presented as mean ± SEM (n = 3 for gene expression; n = 4 for protein expression) with significant differences indicated using asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Expressing, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: ( a ) Gene expression of Tie2, Pecam , and VE-cadherin are shown for WT EBs and VIM −/− EBs over 7 days of differentiation (all normalized to Gapdh ). ( b ) Immunohistochemical analysis of TIE2, PECAM, and VE-CADHERIN protein expression (green) with a nuclear counterstain (blue) in EBs at Days 8 and 10. All images were taken at the same magnification and the scale bar represents 200 μm. ( c ) The bar graphs show the percentage of positive cells for TIE2, PECAM, and VE-CADHERIN at Day 10. Data are presented as mean ± SEM (n = 3 for gene expression; n = 4 for protein expression) with significant differences indicated using asterisks (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Expressing, Immunohistochemical staining