Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: CANu1, a novel nucleolar protein, accumulated on centromere in response to DNA damage.

doi: 10.1111/j.1365-2443.2008.01205.x

Figure Lengend Snippet: Figure 1 Highly conservated human CANu1 protein in Eukaryota. (A) ClustalW mutilple alignment of the deduced amino acid sequences from human c14orf120 (accession no. BC030817) and other specie’s homologous sequences (accession no. NP081166, XP224181, NP608956, NP491806 and NP973785, respectively). Identical residues are shade of black and conserved substitutions are shade of gray. The numbers of right side represent the length of each sequence. (B) This scheme depicts CANu1 domain analyzed by conserved domain database (CDD) from NCBI. The numbers above the schematic structure indicate the length of the amino acids.

Article Snippet: Primary antibody for a mouse homolog of CANu1 NGDN (1 : 100 in dilution; Atlas Antibodies, Cleveland, OH) was treated for 1 h at room temperature.

Techniques: Sequencing

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: CANu1, a novel nucleolar protein, accumulated on centromere in response to DNA damage.

doi: 10.1111/j.1365-2443.2008.01205.x

Figure Lengend Snippet: Figure 2 The expression pattern of CANu1 in cell lines and tissues. (A) RT- PCR analysis for CANu1 was performed. GAPDH was used as a control. (B) Northern blot analysis of CANu1. RNA was isolated from human cancer cell lines and loaded in 1% formaldehyde–agarose gels. Membrane was hybridized with a CANu1 probe and developed using a Molecular Imager FX imaging system. RNA markers are pro- vided at the right. Human β-actin was used as a loading control. (C) The distribution of CANu1 protein in human dorsal skin was analyzed by immunohistochemistry. Arrow head indicates nucleolus.

Article Snippet: Primary antibody for a mouse homolog of CANu1 NGDN (1 : 100 in dilution; Atlas Antibodies, Cleveland, OH) was treated for 1 h at room temperature.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Control, Northern Blot, Isolation, Membrane, Imaging, Immunohistochemistry

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: CANu1, a novel nucleolar protein, accumulated on centromere in response to DNA damage.

doi: 10.1111/j.1365-2443.2008.01205.x

Figure Lengend Snippet: Figure 3 The subcellular localization of CANu1 protein in U2OS cells. (A) U2OS cells were transfected with the pEGFPC1-CANu1 plasmid and were stained by TRITC-conjugated α-nucleolin/C23 antibody to visualize nucleoli. DNA was stained by DAPI. (B) U2OS cells were transiently transfected with plasmids containing CANu1 fragments fused with GFP. Diagram of GFP-CANu1 truncated con- structs and their nucleolar localizations were presented as (+), localized; (–), not localized. Black box indicates Sas10/Utp3 domain and dark grey box indicates GFP.

Article Snippet: Primary antibody for a mouse homolog of CANu1 NGDN (1 : 100 in dilution; Atlas Antibodies, Cleveland, OH) was treated for 1 h at room temperature.

Techniques: Transfection, Plasmid Preparation, Staining

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: CANu1, a novel nucleolar protein, accumulated on centromere in response to DNA damage.

doi: 10.1111/j.1365-2443.2008.01205.x

Figure Lengend Snippet: Figure 4 Knock-down of CANu1 gene in U2OS cells by siRNAs. (A) U2OS cells were transfected with CANu1 siRNAs and scrambled siRNA (as a control). After 72 h, cells were fixed and were analyzed by flow cytometry to determine cell cycle stages. (B) siRNA-delivered cells were harvested and lysed for SDS-PAGE analysis. Samples were loaded to SDS-PAGE gel and analyzed by indicated antibodies. (C) Cells were delivered siRNAs. After 48 h, siRNA treated- cells were allowed expression of GFP-B23. These cells were fixed and observed by fluorescence microscopy.

Article Snippet: Primary antibody for a mouse homolog of CANu1 NGDN (1 : 100 in dilution; Atlas Antibodies, Cleveland, OH) was treated for 1 h at room temperature.

Techniques: Knockdown, Transfection, Control, Flow Cytometry, SDS Page, Expressing, Fluorescence, Microscopy

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: CANu1, a novel nucleolar protein, accumulated on centromere in response to DNA damage.

doi: 10.1111/j.1365-2443.2008.01205.x

Figure Lengend Snippet: Figure 5 Translocation of CANu1 protein in DNA damaged-cells. (A) Cells expressing GFP-B23, GFP-CANu1 and its deletion mutants were irradiated by UV (40 J/m2). Six hours later, these samples were fixed and stained by DAPI. The patterns of localization were observed by fluorescence microscopy. (B) Under UV stress, GFP-CANu1 and HP1α-DsRed expressing cells were observed by confocal microscopy (left column). For HP1β-DsRed, same experiment was conducted as HP1α-DsRed accomplished (right column). (C) Cells expressing GFP-CANu1 protein were irradiated by UV. After 6 h, cells were stained by Cy3-conjugated α-CREST antibody as a centromere marker.

Article Snippet: Primary antibody for a mouse homolog of CANu1 NGDN (1 : 100 in dilution; Atlas Antibodies, Cleveland, OH) was treated for 1 h at room temperature.

Techniques: Translocation Assay, Expressing, Irradiation, Staining, Fluorescence, Microscopy, Confocal Microscopy, Marker

Journal: Genes to cells : devoted to molecular & cellular mechanisms

Article Title: CANu1, a novel nucleolar protein, accumulated on centromere in response to DNA damage.

doi: 10.1111/j.1365-2443.2008.01205.x

Figure Lengend Snippet: Figure 6 The translocation of CANu1 protein in U2OS cells in response to DNA damage agents. GFP-CANu1 expressing cells were treated with SP600125 as a JNK2 inhibitor or caffeine followed by UVC (254 nm, 40 J/m2) or actinomycin D, respectively. Cells were observed by fluo- rescence microscopy.

Article Snippet: Primary antibody for a mouse homolog of CANu1 NGDN (1 : 100 in dilution; Atlas Antibodies, Cleveland, OH) was treated for 1 h at room temperature.

Techniques: Translocation Assay, Expressing, Microscopy

Journal: Nucleic Acids Research

Article Title: Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

doi: 10.1093/nar/gkw790

Figure Lengend Snippet: Identification of NOL10 and NGDN in complex with AATF. ( A ) AATF was isolated by immunoprecipitation (IP) from HeLa cells using an affinity-purified antibody coupled to protein A/G beads. Beads without antibodies were used as negative control. Co-precipitated proteins were analyzed by SDS-PAGE followed by silver staining. Numbering next to the gel refers to MS analysis of excised bands from a Coomassie Blue-stained gel, which identified AATF, NOL10 and NGDN among the most abundant proteins at the AATF complex (Supplementary Table S1). For band 1 to 8, the two proteins with the highest peptide number detected are listed. Bands 9–15 contained predominantly ribosomal proteins. Note that AATF is inefficiently eluted from the antibody. ( B ) Western blot analysis of input (0.04%) and eluate (20%) samples from experiment in (A) confirms the strong enrichment of NOL10 and NGDN in the protein complex isolated by AATF IP. ( C ) Tandem affinity purification (TAP) of NOL10 and NGDN in complex with AATF. Strep-HA (StHA)-tagged NOL10, NGDN or HASt-tagged GFP were induced with tetracycline in HEK293 cell lines and cell extracts (input) subjected to TAP. Inputs (0.008%) and TAP eluates (20%) were analyzed by immunoblotting using the indicated antibodies. Asterisks indicate the StHA-tagged NOL10 bait protein and a degradation product recognized by the anti-NOL10 and anti-HA antibodies. ( D ) HeLa cell extract was separated by centrifugation on a linear 10–45% sucrose gradient. Proteins present in the input and gradient fractions were analyzed by immunoblotting.

Article Snippet: The following antibodies have been described previously: α-ENP1, α-NOB1, α-RPS3 ( ); α-CRM1 ( ); α-RPL23A ( ). α-FBL (sc-166001) and α-NAT10 (sc-271770) were purchased from Santa Cruz Biotechnology; α-NOL10 (ab181161) from Abcam; α-NGDN (STJ24765) from St John's Laboratory; α-actin (A1978) from Sigma and α-HA (ENZ-ABS120) from Enzo Life Sciences.

Techniques: Isolation, Immunoprecipitation, Affinity Purification, Negative Control, SDS Page, Silver Staining, Staining, Western Blot, Centrifugation

Journal: Nucleic Acids Research

Article Title: Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

doi: 10.1093/nar/gkw790

Figure Lengend Snippet: Co-stabilization of AATF, NOL10 and NGDN. ( A ) HeLa cells were transfected with the indicated siRNAs. After 72 h, localization of various factors was analyzed by IF. AATF, NOL10 and NGDN were co-stained with FBL as a nucleolar marker. Scale bar, 20 μm. ( B ) Analysis of RNAi efficiency and protein levels by immunoblotting after 72 h of RNAi. Two independent siRNAs targeting AATF, NOL10 or NGDN and one siRNA for FBL depletion were used.

Article Snippet: The following antibodies have been described previously: α-ENP1, α-NOB1, α-RPS3 ( ); α-CRM1 ( ); α-RPL23A ( ). α-FBL (sc-166001) and α-NAT10 (sc-271770) were purchased from Santa Cruz Biotechnology; α-NOL10 (ab181161) from Abcam; α-NGDN (STJ24765) from St John's Laboratory; α-actin (A1978) from Sigma and α-HA (ENZ-ABS120) from Enzo Life Sciences.

Techniques: Transfection, Staining, Marker, Western Blot

Journal: Nucleic Acids Research

Article Title: Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

doi: 10.1093/nar/gkw790

Figure Lengend Snippet: Mapping of protein interaction domains between AATF, NOL10 and NGDN. ( A ) Scheme of protein domains and secondary structure predictions for AATF, NOL10 and NGDN. ( B–D ) StHA-tagged fl and truncated versions of AATF, NOL10 and NGDN were expressed by transient transfection in HEK293 cells for 48 h. GFP-StHA was used as negative control. StHA-tagged proteins were isolated using StrepTactin sepharose (Strep PD). Protein levels in the inputs (0.45%) and the retrieved protein complexes (30%) were analyzed by immunoblotting using the indicated antibodies.

Article Snippet: The following antibodies have been described previously: α-ENP1, α-NOB1, α-RPS3 ( ); α-CRM1 ( ); α-RPL23A ( ). α-FBL (sc-166001) and α-NAT10 (sc-271770) were purchased from Santa Cruz Biotechnology; α-NOL10 (ab181161) from Abcam; α-NGDN (STJ24765) from St John's Laboratory; α-actin (A1978) from Sigma and α-HA (ENZ-ABS120) from Enzo Life Sciences.

Techniques: Transfection, Negative Control, Isolation, Western Blot

Journal: Nucleic Acids Research

Article Title: Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

doi: 10.1093/nar/gkw790

Figure Lengend Snippet: AATF binds NGDN directly. ( A ) NGDN binds AATF directly via its N-terminal UTP3/SAS10 domain. GST or its fusions with full-length NGDN (fl: 1–315) or fragments (1–148 and 149–315) were co-expressed with MBP-tagged AATF in E. coli . GST-tagged proteins were bound to glutathione beads (GST PD). Input samples (0.04%) and bound fractions (30%) were analyzed by SDS-PAGE followed by Coomassie blue staining. The asterisk indicates an unspecific protein co-purifying with GST. ( B ) AATF residues 208–552 are necessary for efficient binding to NGDN. Full-length and truncated AATF constructs fused to MBP were co-expressed with either GST or GST-tagged NGDN(1–148) in E. coli . Proteins were retrieved from cell extracts by pull-down using glutathione beads. Samples were analyzed as in (A). The asterisk indicates an unspecific protein co-purifying with GST. ( C ) Schematic depiction of domains involved in protein-protein interactions in the ANN complex.

Article Snippet: The following antibodies have been described previously: α-ENP1, α-NOB1, α-RPS3 ( ); α-CRM1 ( ); α-RPL23A ( ). α-FBL (sc-166001) and α-NAT10 (sc-271770) were purchased from Santa Cruz Biotechnology; α-NOL10 (ab181161) from Abcam; α-NGDN (STJ24765) from St John's Laboratory; α-actin (A1978) from Sigma and α-HA (ENZ-ABS120) from Enzo Life Sciences.

Techniques: SDS Page, Staining, Binding Assay, Construct, Protein-Protein interactions

Journal: Nucleic Acids Research

Article Title: Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

doi: 10.1093/nar/gkw790

Figure Lengend Snippet: AATF(208–560) is functionally active and stabilizes NGDN and NOL10 levels in vivo . ( A ) An RNAi-rescue experiment was performed in HeLa cells using the localization of ENP1 after LMB treatment (20 nM, 2 h) as readout for nucleolar steps in ribosome synthesis. siRNA treatment was done for 72 h. 24 h after siRNA delivery, cells were transfected with RNAi-resistant constructs encoding for either HA-tagged full-length AATF (fl) or AATF(208-560). Localization of ENP1 and AATF constructs was visualized by IF. Scale bar, 20 μm. ( B ) Three independent experiments were performed as in (A) and quantified as described in Figure . Mean ± SEM (error bars); n ≥ 58; *** P ≤ 0.001 (unpaired t-test). AATF-HA (fl) and AATF(208-560)-HA significantly complement the AATF depletion effect on nucleolar ribosome biogenesis. ( C ) RNAi and transfection were performed as in (A), including the AATF(455–560) truncated version. Rescue of NGDN and NOL10 levels was analyzed by IF and nuclei were stained with Hoechst.

Article Snippet: The following antibodies have been described previously: α-ENP1, α-NOB1, α-RPS3 ( ); α-CRM1 ( ); α-RPL23A ( ). α-FBL (sc-166001) and α-NAT10 (sc-271770) were purchased from Santa Cruz Biotechnology; α-NOL10 (ab181161) from Abcam; α-NGDN (STJ24765) from St John's Laboratory; α-actin (A1978) from Sigma and α-HA (ENZ-ABS120) from Enzo Life Sciences.

Techniques: In Vivo, Transfection, Construct, Staining

Journal: Nucleic Acids Research

Article Title: Human AATF/Che-1 forms a nucleolar protein complex with NGDN and NOL10 required for 40S ribosomal subunit synthesis

doi: 10.1093/nar/gkw790

Figure Lengend Snippet: AATF, NOL10 and NGDN depletion causes pre-rRNA processing defects at cleavage sites A0, 1 and in the ITS1 region. ( A ) Indicated proteins were depleted by RNAi treatment for 72 h in HeLa cells. ENP1 localization was visualized by IF analysis after 2 h of LMB treatment (20 nM). Scale bar, 20 μm. ( B ) Northern blot analysis of total RNA extracted from HeLa cells after siRNA treatment for 72 h. Radioactively labeled probes binding to the 5′ITS1, 5′ETS, ITS2 or actin mRNA were used to detect the indicated rRNA precursors or mRNA. Mature 18S and 28S rRNA were visualized by GelRed staining on the membrane. ( C ) Quantification of three independent experiments using the 5′ITS1 probe as shown in (B) using ImageJ. The ‘47S’ (47S, 45S and 43S pre-rRNAs) pre-rRNA signal intensity was used for normalization. Mean ± SD (error bars). ( D ) Scheme illustrating pre-rRNA processing defects observed in (B). Depletion of AATF, NOL10 and NGDN leads to increased levels of 30S and reduced levels of 41S pre-rRNA, indicating lower efficiency in cleavage of 18S pre-rRNA at the sites A0, 1 and in the ITS1 region. ( E ) HeLa cells were subjected to RNAi for 48 h. Processing of newly synthesized pre-rRNA was analyzed after 33 P pulse-labeling of HeLa cells. At the end of the indicated chase periods, RNA was extracted and separated by gel electrophoresis. Total mature 18S and 28S rRNA were stained with GelRed. ( F ) Quantification of three independent pulse-labeling experiments as shown in Supplementary Figure S5D. FBL, AATF, NOL10 and NGDN were depleted by siRNA treatment for 48 h and newly synthesized mature 18S and 28S rRNA were quantified after a chase period of 4 h. Mean ± SD (error bars).

Article Snippet: The following antibodies have been described previously: α-ENP1, α-NOB1, α-RPS3 ( ); α-CRM1 ( ); α-RPL23A ( ). α-FBL (sc-166001) and α-NAT10 (sc-271770) were purchased from Santa Cruz Biotechnology; α-NOL10 (ab181161) from Abcam; α-NGDN (STJ24765) from St John's Laboratory; α-actin (A1978) from Sigma and α-HA (ENZ-ABS120) from Enzo Life Sciences.

Techniques: Northern Blot, Labeling, Binding Assay, Staining, Membrane, Synthesized, Nucleic Acid Electrophoresis