Journal: Genome Research

Article Title: Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation

doi: 10.1101/gr.154872.113

Figure Lengend Snippet: Chromatin profiles of SUMO1 and SUMO2. ( A ) Association of SUMO1 ( left ) and SUMO2 ( right ) with Pol II, H3K4me3, H3K27me3, and H3K9me3 in WI38 cells. Comparison of tag density in the region of ±5 kb around the SUMO-occupied loci. Clustering identifies five classes as indicated. ( B ) A pie chart representation of the distribution of SUMO sites in five different genomic regions. The definition of each region is described below . ( C ) Frequency of SUMO1 and SUMO2 site localization with respect to TSS. ( D ) Comparison of SUMO2 tag density around the SUMO1 peaks ( left ) and of SUMO1 tag density around the SUMO2 peaks ( right ). ( E ) Histogram representing the percentage of the H3K4me3, H3K27me3, or H3K9me3 peaks with an overlapping SUMO1 and/or SUMO2 peak.

Article Snippet: Culturing of human diploid WI38 fibroblasts (ATCC) and infection by retroviral-mediated gene transfer with either pBABE-puro-HRAS G12V , pBABE-puro-HA-PIASY, or empty vector as a control were performed at a physiological oxygen concentration of 3% as previously described ( ) or at 21% for growth analysis.

Techniques: Comparison

Journal: Genome Research

Article Title: Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation

doi: 10.1101/gr.154872.113

Figure Lengend Snippet: Chromatin profiles of UBC9 and PIASY. ( A ) Association of UBC9 ( left ) and PIASY ( right ) with SUMO1, SUMO2, Pol II, H3K4me3, H3K27me3, and H3K9me3 in WI38 proliferating cells. Comparison of the tag density in the ±5-kb region around UBC9- or PIASY-bound loci. Clustering identifies four classes as indicated. ( B ) Pie chart representation of UBC9 and PIASY binding site distribution in five different genomic regions as described in . ( C ) Venn diagram representing overlap between SUMO1- and/or SUMO2-, UBC9- and PIASY-marked TSS. ( D ) Merged profiles of the SUMO machinery read density with respect to distance from TSS. ( E,F ) A Genome Browser view of the indicated ChIP-seq data at the FOS ( E ) and HRAS loci ( F ). ( G ) DAVID functional annotation of TSS marked by SUMO, UBC9, and PIASY ( left ) or SUMO, UBC9, but no PIASY ( right ). The top overrepresented categories are shown.

Article Snippet: Culturing of human diploid WI38 fibroblasts (ATCC) and infection by retroviral-mediated gene transfer with either pBABE-puro-HRAS G12V , pBABE-puro-HA-PIASY, or empty vector as a control were performed at a physiological oxygen concentration of 3% as previously described ( ) or at 21% for growth analysis.

Techniques: Comparison, Binding Assay, ChIP-sequencing, Functional Assay

Journal: Genome Research

Article Title: Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation

doi: 10.1101/gr.154872.113

Figure Lengend Snippet: Sumoylation controls expression of histone and growth control genes. ( A ) WI38 cells were infected with lentiviruses expressing control shCt or shUBC9 shRNAs. Five days post-selection, the expression of the indicated genes was analyzed by RT-qPCR. ( B ) Western blot analysis of WI38 cells expressing shCt or shUBC9 showing expression of histones H3, H2A, H2B, and H4. Tubulin and Ponceau were used as loading controls. Depletion of UBC9 and concomitant loss of global sumoylation and sumoylated SP100 are shown as controls for knockdown efficiency. ( C ) WI38 cells were transfected with a control siCt or siPIASY siRNAs, and the expression of the indicated genes was analyzed by RT-qPCR. ( D ) Affymetrix analysis of histone mRNA differential expression in retrovirally infected WI38 cells overexpressing PIASY or a control vector (WT). ( E–G ) As in A . ( H ) Forward scatter analysis (FSC) of Ubc9 +/+ (WT) and Ubc9 fl/-;T2 (KO) MEFs treated for 7 d by tamoxifen (four embryos/genotype); mean size (FSC units): 628 ± 17.5 ( Ubc9 −/− ) versus 594.3 ± 7.1 ( Ubc9 +/+ ); P -value = 0.006; one representative example is shown. ( I ) Total protein levels as measured by OD normalized to cell number of Ubc9 +/+ (WT) and Ubc9 fl/-;T2 (KO) MEFs treated for 7 d by tamoxifen (four embryos/genotype); mean amount (μg/mL): 219.3 ± 32.9 ( Ubc9 −/− ) versus 193.5 ± 63.2 ( Ubc9 +/+ ); P -value = 0.019. ( J ) As in A . For all RT-qPCR, experiments were carried out in triplicate and data are represented as mean ±SEM ( n = 3).

Article Snippet: Culturing of human diploid WI38 fibroblasts (ATCC) and infection by retroviral-mediated gene transfer with either pBABE-puro-HRAS G12V , pBABE-puro-HA-PIASY, or empty vector as a control were performed at a physiological oxygen concentration of 3% as previously described ( ) or at 21% for growth analysis.

Techniques: Expressing, Control, Infection, Selection, Quantitative RT-PCR, Western Blot, Knockdown, Transfection, Quantitative Proteomics, Plasmid Preparation

Journal: Genome Research

Article Title: Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation

doi: 10.1101/gr.154872.113

Figure Lengend Snippet: Depletion of UBC9 induces altered gene expression program together with a senescence-related phenotype. ( A,B ) Top selected categories identified by DAVID ontology analysis of up-regulated ( A ) and down-regulated ( B ) genes marked by SUMO1 and/or SUMO2 in their promoters in shUBC9 WI38 cells. ( C ) Growth curve of WI38 cells following infection with lentiviruses expressing shCt or shUBC9 shRNAs. ( D ) Percentage of EdU and SA-β-Gal positive WI38 cells at 4 d (early) and 8 d (late) post-infection. ( E ) Representative micrographs showing SA-β-Gal staining. ( F ) Western blot analysis of shCt or shUBC9 WI38 extracts for the indicated senescence markers. Actin and GAPDH were used as loading controls. ( G ) WI38 cells infected with lentiviruses expressing shCt, shUBC9, GFP, or HRAS G12V were stained with propidium iodide and subjected to cell cycle analysis by flow cytometry. ( H ) Venn diagram showing overlap between Affymetrix gene expression profiles of HRAS G12V -induced senescent and shUBC9 WI38 cells. Heat map below represents fold changes (FC) of selected genes in the two data sets. ( I ) Immunostaining of WI38 infected cells with control shCt, shUBC9, or HRAS G12V as indicated and stained for PML (red), H3K9me3 (green), and DAPI for SAHF visualization. The unique PML aggregate in the shUBC9 cells is used as a positive control for knockdown efficiency . ( Right ) Graph presenting the associated percentages of SAHF positive nuclei.

Article Snippet: Culturing of human diploid WI38 fibroblasts (ATCC) and infection by retroviral-mediated gene transfer with either pBABE-puro-HRAS G12V , pBABE-puro-HA-PIASY, or empty vector as a control were performed at a physiological oxygen concentration of 3% as previously described ( ) or at 21% for growth analysis.

Techniques: Gene Expression, Infection, Expressing, Staining, Western Blot, Cell Cycle Assay, Flow Cytometry, Immunostaining, Control, Positive Control, Knockdown

Journal: Genome Research

Article Title: Sumoylation at chromatin governs coordinated repression of a transcriptional program essential for cell growth and proliferation

doi: 10.1101/gr.154872.113

Figure Lengend Snippet: The SUMO machinery is released from chromatin in senescent cells. ( A ) Comparison of SUMO1 association with Pol II, H3K4me3, H3K27me3, and H3K9me3 in proliferating (P) and HRAS G12V -induced senescent (S) WI38 cells. ( B ) ChIP-qPCR for SUMO1 in proliferating (P), HRAS G12V -induced senescent (RAS), and replicative senescent (RS) WI38 cells on MTBP/MRPL13 and FOS promoters. ( C ) Proliferating and senescent cells were fractionated, and the presence of the indicated proteins in each fraction was quantitated by Western blot. Equal amounts of proteins were loaded for total lysate, Cyt (cytoplasmic fraction), SNE (soluble nuclear extract), and pellet (insoluble fraction). ( D ) Plots showing peak density in proliferating (red) and senescent (blue) cells over chromosome 6. ( E ) Scatter plot comparison of differential gene expression as measured by RNA-seq and differential occupancy of SUMO1 ( left ) or SUMO2 ( right ) (peak height fold change) in proliferating versus senescent cells. Differential expression is shown as log 2 of reads per RPKM.

Article Snippet: Culturing of human diploid WI38 fibroblasts (ATCC) and infection by retroviral-mediated gene transfer with either pBABE-puro-HRAS G12V , pBABE-puro-HA-PIASY, or empty vector as a control were performed at a physiological oxygen concentration of 3% as previously described ( ) or at 21% for growth analysis.

Techniques: Comparison, ChIP-qPCR, Western Blot, Gene Expression, RNA Sequencing, Quantitative Proteomics

Journal: Mechanisms of Ageing and Development

Article Title: High-affinity Na + -dependent dicarboxylate cotransporter promotes cellular senescence by inhibiting SIRT1

doi: 10.1016/j.mad.2010.08.006

Figure Lengend Snippet: NaDC3-induced senescence-related phenotypes in WI38 cells. (A) Efficiency of infection with NaDC3 was detected by Western blot. The WI38/hNaDC3 cells showed significantly high level of NaDC3, but no expression was found in control groups ( P < 0.05) ( n = 6). Empty vector groups had no difference compared to control ( P > 0.05) ( n = 6). Relative NaDC3 protein band density was normalized to β-actin. * P < 0.05 versus control group. (B) Morphology observation in the WI38 cells infected with NaDC3 vector. WI38/hNaDC3, WI38/neo and WI38/con cells cultured for 37PD were observed for morphology. (C) SA-beta-gal staining in the WI38 cells infected with NaDC3 retroviral vector. WI38 cells cultured for 37PD were stained for SA-beta-gal, which showed blue precipitation in the cytoplasm in senescent cells. Magnification, 100×. (D) Analysis of SAHF formation. The cells were stained with DAPI and heterochromatin foci were shown in the senescent cells. (E) Analysis of cell cycle by flow cytometry.

Article Snippet: Human embryonic lung diploid fibroblasts WI38 and MRC-5 were obtained from ATCC.

Techniques: Infection, Western Blot, Expressing, Control, Plasmid Preparation, Cell Culture, Staining, Retroviral, Flow Cytometry

Journal: Mechanisms of Ageing and Development

Article Title: High-affinity Na + -dependent dicarboxylate cotransporter promotes cellular senescence by inhibiting SIRT1

doi: 10.1016/j.mad.2010.08.006

Figure Lengend Snippet: SIRT1 inhibited Senescence-related phenotypes in WI38 cells. The SA-beta-gal staining and DAPI staining in WI38 cells were observed. The SA-beta-gal activity (A) and SAHF formation (B) were observed in the WI38 cells treated with resveratrol or niacinamide.

Article Snippet: Human embryonic lung diploid fibroblasts WI38 and MRC-5 were obtained from ATCC.

Techniques: Staining, Activity Assay

Journal: Mechanisms of Ageing and Development

Article Title: High-affinity Na + -dependent dicarboxylate cotransporter promotes cellular senescence by inhibiting SIRT1

doi: 10.1016/j.mad.2010.08.006

Figure Lengend Snippet: NaDC3 inhibited SIRT1 Activity in WI38 cells. SIRT1 activity of WI38/hNaDC3 cells was inhibited dramatically, compared with those in control and WI38/neo groups ( P < 0.05). * P < 0.05 versus control group ( n = 6).

Article Snippet: Human embryonic lung diploid fibroblasts WI38 and MRC-5 were obtained from ATCC.

Techniques: Activity Assay, Control

Journal: Mechanisms of Ageing and Development

Article Title: High-affinity Na + -dependent dicarboxylate cotransporter promotes cellular senescence by inhibiting SIRT1

doi: 10.1016/j.mad.2010.08.006

Figure Lengend Snippet: Elevation of SIRT1 activity reduced senescence-related phenotypes induced by NaDC3. (A) SIRT1 activity was measured using the Fluor de Lys kit in WI38/hNaDC3 cells in the presence of CR serum or resveratrol cultured for 37PD. WI38/hNaDC3 cells in the presence of CR serum or resveratrol showed significantly higher levels of SIRT1 than that in WI38/hNaDC3 group ( P < 0.05). * P < 0.05 versus WI38/hNaDC3 group ( n = 6). (B) Effect of elevating SIRT1 activity on morphology and SA-beta-gal staining of WI38/hNaDC3 cells. WI38/hNaDC3 cells cultured in the presence CR serum or resveratrol for 37 PD were stained for SA-beta-gal. The percentage of SA-beta-gal stained positive cells. For each group, 100–200 cells were counted. Comparison among groups was conducted with ANOVA. * P < 0.05 versus WI38/hNaDC3 group. Magnification, 100×. (C) SAHF formations were observe in the WI38/hNaDC3 cells treated with resveratrol or CR serum. (D) Analysis of cell cycle by flow cytometry in the WI38/hNaDC3 cells treated with resveratrol or CR serum.

Article Snippet: Human embryonic lung diploid fibroblasts WI38 and MRC-5 were obtained from ATCC.

Techniques: Activity Assay, Cell Culture, Staining, Comparison, Flow Cytometry

Journal: Mechanisms of Ageing and Development

Article Title: High-affinity Na + -dependent dicarboxylate cotransporter promotes cellular senescence by inhibiting SIRT1

doi: 10.1016/j.mad.2010.08.006

Figure Lengend Snippet: Exogenous NAD + delayed cellular premature senescence induced by NaDC3. (A) NAD + /NADH ratio of WI38/hNaDC3 cells was down-regulated. But WI38/hNaDC3 + NAD cells were noted NAD + /NADH ratio was enhanced, compared with WI38/hNaDC3 cells in 37 PD ( P < 0.05). * P < 0.05 versus WI38/hNaDC3 group. (B) SIRT1 activity was measured in WI38/hNaDC3 + NAD + cells. NAD + enhanced SIRT1 activity of WI38/hNaDC3 cells, compared with WI38/hNaDC3 cells without NAD + in 37 PD ( P < 0.05). * P < 0.05 versus WI38/hNaDC3 group. The data shown is one representative experiment out of a series of five with similar results. (C) The change of morphology and the percentage of SA-beta-gal stained positive cells. For each group in 37 PD, 100–200 cells were counted. Comparison among groups was conducted with ANOVA. * P < 0.05 versus WI38/hNaDC3 group. Magnification, 100×. (D) SAHF formations were observed in the WI38/hNaDC3 cells treated with NAD + .

Article Snippet: Human embryonic lung diploid fibroblasts WI38 and MRC-5 were obtained from ATCC.

Techniques: Activity Assay, Staining, Comparison

Journal: Mechanisms of Ageing and Development

Article Title: High-affinity Na + -dependent dicarboxylate cotransporter promotes cellular senescence by inhibiting SIRT1

doi: 10.1016/j.mad.2010.08.006

Figure Lengend Snippet: A proposed signaling pathway involved in NaDC3-induced aging in the WI38 cells.

Article Snippet: Human embryonic lung diploid fibroblasts WI38 and MRC-5 were obtained from ATCC.

Techniques: