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First published online 1 September 2005
doi: 10.1242/jcs.02556

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The human Rothmund-Thomson syndrome gene product, RECQL4, localizes to distinct nuclear foci that coincide with proteins involved in the maintenance of genome stability

Maja Petkovic¹, Tobias Dietschy¹, Raimundo Freire², Renjie Jiao^1,* and Igor Stagljar^1,,

¹ Institute of Vet. Biochemistry and Molecular Biology, University of Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland
² Unidad de investigacion, Hospital Universitario de Canarias, Ofra s/n, La Cuesta, 38071 Tenerife, Spain

View larger version (27K): [in a new window]   Fig. 1. Characterization of anti-RecQL4 antibodies. (A) Western blot using the anti-RECQL4 N-t and anti-RECQL4 C-t against HeLa total cell extract (TCE), HeLa cytosolic extracts (CE), HeLa nuclear extract (NE), and nuclear extracts from AG18371 fibroblasts derived from a Rothmund-Thomson-syndrome patient. Control western blots against HeLa CE and HeLa NE were performed with anti-PARP1, anti-BLM and anti-GRB2 antibodies, respectively. (B) Lysates of 293T cells transiently transfected with a vector expressing FLAG-RECQL4 or with the corresponding empty vector were subjected to western blot with anti-FLAG and anti-RECQL4 C-t antibodies. Asterisks indicate breakdown products.

View larger version (58K): [in a new window]   Fig. 2. RECQL4 localizes to nuclear foci in different cell lines. The foci were detected with anti-RECQL4 N-t antibody (red). DAPI-staining shows nuclear DNA. Bar, 10 µm. (A) HeLa cell. (B) WI38/VA13 cell. (C) Primary skin fibroblast. (D) Preimmune serum, HeLa cell.

View larger version (39K): [in a new window]   Fig. 3. Determination of the specificity of the RECQL4 antibodies by siRNA. (A) Western blot with anti-RECQL4 C-t antibody (upper panel) or anti-tubulin antibody (lower panel) using total cell extracts from HeLa cells transfected with 200 nM RECQL4-1 or control-1 siRNA. Asterisks indicate breakdown products. (B) RECQL4 knock-down leads to a loss of RECQL4 nuclear foci. Immunofluorescence analysis with anti-RECQL4 N-t antibody (red) on exponentially growing HeLa cells transfected with 200 nM RECQL4-1 or control-1 siRNA, DAPI-staining shows nuclear DNA (blue). Bar, 10 µm. (C) The mean number of RECQL4 foci per cell after siRNA treatment is displayed in histograms (standard error of the mean is indicated in the columns).

View larger version (27K): [in a new window]   Fig. 4. RECQL4 colocalizes with PML. HeLa cells were stained with anti-RECQL4 (red) and anti-PML (green) antibodies. Where red and green signals overlap, a yellow pattern is seen, indicating colocalization of RECQL4 and PML. DAPI-staining shows nuclear DNA. Bar, 10 µm. (Upper panel) Colocalization of RECQL4 and PML in untreated HeLa cells. (Lower panel) Colocalization of RECQL4 and PML after 10 µM etoposide treatment of HeLa cells.

View larger version (42K): [in a new window]   Fig. 5. RECQL4 is associated with regions of ssDNA and colocalizes with Rad51, but it does not colocalize with BRCA1. (A) HeLa cells were grown in BrdU-containing medium and stained for RECQL4 (red), BrdU (green) and nuclear DNA (blue). Bar, 10 µm. (Upper panel) RecQL4 and BrdU staining in untreated cells. (Lower panel) RECQL4 colocalizes with ssDNA after treatment with 10 µM etoposide. (B) RECQL4 colocalizes with Rad51. WI38/VA13 cells were stained with anti-RECQL4 (red) and anti-Rad51 antibodies (green), and DAPI (blue). RECQL4 and Rad51 antibody-staining in untreated cells (upper panel) and after 10 µm etoposide treatment (lower panel). Bar, 10 µm. (C) RECQL4 and Rad51 form a complex in human cells. 293T cells were transiently transfected with FLAG-RECQL4, nuclear extracts derived from these cells were immunoprecipitated with the anti-FLAG antibody, and analysed by SDS-polyacrylamide gel electrophoresis. One-tenth of the same nuclear extract was used as input control (lane 1). Immunoprecipitated FLAG-RECQL4 (upper and middle panels) and Rad51 (lower panel) were detected by western blotting with the anti-FLAG, anti-RECQL4 C-t and anti-Rad51 antibodies, but not with the control IgG (lane 2). Reciprocal co-immunoprecipitation is shown in lanes 4-6; lane 4, input; lane 5, immunoprecipitation with the control IgG; lane 6, immunoprecipitation with an anti-Rad51 antibody. (D) Colocalization of RECQL4 with BRCA1 in untreated HeLa cells (upper panel) and in HeLa cells treated with 10 µM etoposide (lower panel). RECQL4 (red), BRCA1 (green), DAPI-staining shows nuclear DNA. Bar, 10 µm.

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