donson cdna clone sc111799  (OriGene)

Journal: Molecular Systems Biology

Article Title: Clustering phenotype populations by genome-wide RNAi and multiparametric imaging

doi: 10.1038/msb.2010.25

Figure Lengend Snippet: Functional analysis of candidate genes for roles in cell-cycle progression and spindle organization. ( A ) Time-resolved cell-cycle analysis in HeLa cells transfected with indicated siRNAs at different time points. Box colours represent fractions of cells with DNA content corresponding to sub-G1, G1/G0, S and G2/M. HeLa cells transfected with siRNAs against DONSON showed a delay in S-phase progression. ( B ) Assessment of S-phase progression by BrdU incorporation; 48 h after siRNA transfection, U2OS cells were synchronized for 16 h with 1 mM hydroxyurea (HU) and released for 6 h in BrdU-containing medium. BrdU-positive cells were stained with anti-BrdU primary antibody (Calbiochem) and Alexa 488 secondary antibody. DNA was counterstained with propidium iodide. Imaging and quantification were performed with Acumen Explorer microplate reader. Values are shown as mean±s.d. of three biological replicates. ( C ) Cell-cycle-dependent protein expression of DONSON. U2OS cells were synchronized either in G1/S with HU or in G2/M with nocodazole (Noc). Cells were collected at different time points after release into cell cycle for western blot analysis. ( D ) DONSON depletion is associated with a 10-fold increase in multipolar spindles compared to control treatments. U2OS cells were transfected with a DONSON siRNA pool and immunostained for α- and γ-tubulin at indicated time points. U2OS cells transfected with Rluc siRNAs serving as negative control. Data represent mean±s.d. of three biological replicates. At least 200 metaphase spindles were counted in each experiment. Scale bar indicates 2.5 μm. ( E ) DONSON protein co-localizes with centrosomes. HeLa cells were transfected with HA-tagged DONSON for 48 h and immunostained with primary HA antibody and Alexa 488-conjugated secondary antibody. Arrows indicate the centrosomal staining of DONSON (green). DNA was counterstained with DAPI (blue). Scale bar indicates 2.5 μm. ( F ) DONSON co-localizes with centrin. U2OS cells were transfected with HA-tagged DONSON; 48 h after transfection, cells were immunostained with anti-centrin and anti-HA-tagged primary antibodies and Alexa 488-, Alexa 594-conjugated secondary antibodies, respectively. Scale bar indicates 2.5 μm. Source data is available for this figure at www.nature.com/msb .

Article Snippet: An HA-tagged version of human DONSON was generated by modification of a DONSON cDNA clone (SC111799, OriGene).

Techniques: Functional Assay, Cell Cycle Assay, Transfection, BrdU Incorporation Assay, Staining, Imaging, Expressing, Western Blot, Control, Negative Control

Journal: Molecular Systems Biology

Article Title: Clustering phenotype populations by genome-wide RNAi and multiparametric imaging

doi: 10.1038/msb.2010.25

Figure Lengend Snippet: Functional assays for candidate genes in maintenance of genomic integrity. ( A ) Depletion of RRM1, CLSPN, CD3EAP, CADM1, DONSON and SON induced γH2AX foci formation. U2OS cells were transfected with siRNA pools and immunostained with γH2AX antibody 72 h after transfection. Representative images of γH2AX foci formation are shown. Scale bar indicates 2.5 μm. ( B ) Quantification of γH2AX foci formation on depletion of candidate genes; 72 h after siRNA transfection, U2OS cells were fixed, immunostained for γH2AX and γH2AX-positive cells were quantified. Ratios of γH2AX-positive cells were normalized to the negative control Rluc siRNA treatment. Data are mean±s.d. of three biological replicates. ( C ) γH2AX accumulation in DONSON and SON-depleted cells is not caused by an accumulation of S-phase cells. U2OS cells were collected 72 h after siRNA transfection and cell lysates were analysed by western blotting using indicated antibodies. Source data is available for this figure at www.nature.com/msb .

Article Snippet: An HA-tagged version of human DONSON was generated by modification of a DONSON cDNA clone (SC111799, OriGene).

Techniques: Functional Assay, Transfection, Negative Control, Western Blot

Journal: Molecular Systems Biology

Article Title: Clustering phenotype populations by genome-wide RNAi and multiparametric imaging

doi: 10.1038/msb.2010.25

Figure Lengend Snippet: SON, DONSON and CD3EAP are required for the DNA damage response. ( A ) SON, DONSON and CD3EAP depletion leads to a decreased phosphorylation of CHEK1 on γ irradiation, similar to ATR; 48 h after siRNA transfection, cells were γ irradiated and collected 1 or 2 h later for immunoblot analysis, probing with indicated antibodies. ( B ) SON and DONSON depletion leads to attenuated phosphorylation of CHEK1 on UV exposure, similar to ATR depletion. U2OS cells were UVC irradiated (20 J/m 2 ) 48 h after siRNA transfection. Cell lysates were collected for immunoblotting 2 h later and probed with indicated antibodies. ( C ) Knock down of DONSON and SON impairs RPA2 recruitment onto chromatin and the phosphorylation of ATR substrates. U2OS cells were transfected with siRNAs and UVC irradiated (20 J/m 2 ). Subsequently, the chromatin-associated insoluble fraction was extracted 2 h after UV exposure, and the fractions were analysed for the indicated proteins by immunoblot.

Article Snippet: An HA-tagged version of human DONSON was generated by modification of a DONSON cDNA clone (SC111799, OriGene).

Techniques: Phospho-proteomics, Irradiation, Transfection, Western Blot, Knockdown