clone 4b8  (Cell Signaling Technology Inc)

Journal: Cancer research

Article Title: Transcriptional regulator CNOT3 defines an aggressive colorectal cancer subtype

doi: 10.1158/0008-5472.CAN-16-1346

Figure Lengend Snippet: (A) Schema of the contrast in chromatin at inactive genomic regions (left) and marked nucleosome pairs flanking active, transcription factor (TF)-bound sites (right). (B) Integrative Genome Viewer (IGV) tracks showing H3K4me2+ nucleosomes shared across samples (e.g., at the ACVR1B locus) and others found specifically in certain Ad-Ca pairs (e.g., upstream of ANKRD33). (C) Heatmap representation of unsupervised clustering and Spearman correlations among genome-wide H3K4me2-marked nucleosomes in 10 pairs of human colon cancers (Ca) and the corresponding adenomas (Ad). Correlations are highest between individual Ad-Ca pairs. (D) Heatmap of nucleosomal H3K4me2 signals at the 1000 most differentially marked regions in Ca2 and Ca9, compared to their respective Ad. The sequence motifs most enriched in these Ca, shown below, are attributed to the TFs CNOT3 and TRIM28. (E) IGV traces of representative differential nucleosome pairs in Ad-Ca2 and Ad-Ca9. (F) Distribution of the 1000 top-scoring differential nucleosome pairs in Ca2 and Ca9, compared to their respective precursor Ad.

Article Snippet: Immunodetection For immunohistochemistry, we used Ab against CNOT3 (Novus Biologicals, clone 4B8, 1:50), TRIM28 (Abnova, clone 4E6, 1:250), KI67 (DAKO, clone MIB-1, 1:50) or CTNNB1 (BD Transduction Laboratories, clone 14/Beta-Catenin, 1:250) diluted in Tris-buffered saline (TBS, pH 8.0) containing 1% bovine serum albumin.

Techniques: Genome Wide, Sequencing

Journal: Cancer research

Article Title: Transcriptional regulator CNOT3 defines an aggressive colorectal cancer subtype

doi: 10.1158/0008-5472.CAN-16-1346

Figure Lengend Snippet: (A–C) Immunohistochemistry (IHC) for TRIM28 (A), CNOT3 (B) and KI67 (right) in serial sections of a representative CRC specimen. While TRIM28 is expressed in all tumor cells, CNOT3 is expressed in a minority of scattered nuclei, compared to a much larger fraction of KI67+ cells, with modest overlap of CNOT3 and KI67. The areas boxed in b and c are magnified below. (D) Mean counts (± SD) of CNOT3+ and KI67+ nuclei from 500 cells in each of 3 independent CNOT3+ CRCs. (E) IHC for CNOT3 (left) and KI67 (right) in serial sections of normal human colon mucosa, showing CNOT3 in some (black arrows) but not most (red arrows, e.g.) KI67+ crypt epithelial cells. (F) Immunoblot analysis of CNOT3 in murine intestinal Lgr5+ cells sorted by flow cytometry and in isolated intestinal crypts and villi. HCT116 human CRC cells serve as a positive control. (G) Immunoblot analysis of cytoplasmic (Cy) and nuclear (Nu) fractions of HCT116 and Caco-2 cells, showing presence of CNOT1 and CNOT3 in both fractions. LaminB1 and GAPDH serve as positive controls for the Nu and Cy fractions, respectively. (H) CNOT3 immunocytochemistry in HCT116 and Caco-2 cells, showing both Nu and Cy staining. All scale bars, 50 µm.

Article Snippet: Immunodetection For immunohistochemistry, we used Ab against CNOT3 (Novus Biologicals, clone 4B8, 1:50), TRIM28 (Abnova, clone 4E6, 1:250), KI67 (DAKO, clone MIB-1, 1:50) or CTNNB1 (BD Transduction Laboratories, clone 14/Beta-Catenin, 1:250) diluted in Tris-buffered saline (TBS, pH 8.0) containing 1% bovine serum albumin.

Techniques: Immunohistochemistry, Western Blot, Flow Cytometry, Isolation, Positive Control, Immunocytochemistry, Staining

Journal: Cancer research

Article Title: Transcriptional regulator CNOT3 defines an aggressive colorectal cancer subtype

doi: 10.1158/0008-5472.CAN-16-1346

Figure Lengend Snippet: (A) CNOT3 immunocytochemistry in HCT116 cells treated with control, non-specific (NS) shRNA (left) or with CNOT3-specific shRNA #3 (right), confirming CNOT3 loss and Ab specificity and showing typical changes in morphology of CNOT3-depleted cells. (B) Immunoblot analysis of nuclear (Nu) and cytoplasmic (Cy) fractions of HCT116 cells treated with non-specific (NS) or CNOT3-specific shRNA #3. LaminB1 and GAPDH serve to mark the Nu and Cy fractions, respectively. (C) Reduced CNOT3 levels, achieved with two independent shRNAs (#3 and #5, immunoblot in the inset), impaired proliferation of CRC cell lines HCT116 (left) and Caco-2 (right), compared to treatment with a non-specific (NS) control shRNA. Each value represents the mean ± SD optical density from triplicate samples; other cell lines are shown in Suppl. Fig. 3C. (D) Reduced growth of CNOT3-depleted HCT116 cells in murine xenografts. Each plotted value represents the mean ± SD from 5 replicates. (E–G) Significantly reduced phospho-Histone H3 staining (E, determined manually in blind counts) and BrdU uptake (F, determined by flow cytometry) in CNOT3-depleted HCT116 and Caco-2 cells, without concomitant change in apoptotic AnnexinV+ cells (G, flow cytometry). Graphs represent the mean ± SD from 3 replicates. (F) An shRNA-resistant CNOT3 cDNA construct, but not a GFP cDNA control, restored cell replication in HCT116 cells depleted of endogenous CNOT3 using shRNA #3. The immunoblot shows relative CNOT3 and GAPDH levels in total cell lysates.

Article Snippet: Immunodetection For immunohistochemistry, we used Ab against CNOT3 (Novus Biologicals, clone 4B8, 1:50), TRIM28 (Abnova, clone 4E6, 1:250), KI67 (DAKO, clone MIB-1, 1:50) or CTNNB1 (BD Transduction Laboratories, clone 14/Beta-Catenin, 1:250) diluted in Tris-buffered saline (TBS, pH 8.0) containing 1% bovine serum albumin.

Techniques: Immunocytochemistry, Control, shRNA, Western Blot, Staining, Flow Cytometry, Construct

Journal: Cancer research

Article Title: Transcriptional regulator CNOT3 defines an aggressive colorectal cancer subtype

doi: 10.1158/0008-5472.CAN-16-1346

Figure Lengend Snippet: (A) Representative IGV traces of CNOT3 binding sites identified by ChIP-seq at promoters and intergenic regions in Ca9, Ca2 and HCT116 and corresponding motif analysis and z-scores. (B) Distribution of CNOT3 binding sites in Ca9, Ca2, and HCT116. (C) Overlap of CNOT3 binding sites in the 3 sources of data at TSSs (top) and intergenic regions (bottom). (D) Heatmap of H3K4me2-marked nucleosomes in three CRCs at CNOT3 binding sites identified in Ca9. K-means clustering (k=3) reveals groups of sites with or without marked flanking nucleosomes; IGV traces representing the first cluster and the two other largely similar clusters are shown to the right. (E) Average distribution of H3K4me2 signals in an archival CNOT3hi CRC (inset: CNOT3 IHC; scale bar, 50 µm) flanking the 1,000 most accessible regions in Ca9. (F) GREAT analysis of CNOT3 binding sites ≤30 kb from TSSs, showing the top over-represented categories from the MSigDB ontology of genetic and chemical perturbations. Binomial corrected (FDR) p-values are shown at log10 scale; all GREAT-derived ontologies are listed in Suppl. Table 2.

Article Snippet: Immunodetection For immunohistochemistry, we used Ab against CNOT3 (Novus Biologicals, clone 4B8, 1:50), TRIM28 (Abnova, clone 4E6, 1:250), KI67 (DAKO, clone MIB-1, 1:50) or CTNNB1 (BD Transduction Laboratories, clone 14/Beta-Catenin, 1:250) diluted in Tris-buffered saline (TBS, pH 8.0) containing 1% bovine serum albumin.

Techniques: Binding Assay, ChIP-sequencing, Derivative Assay

Journal: Cancer research

Article Title: Transcriptional regulator CNOT3 defines an aggressive colorectal cancer subtype

doi: 10.1158/0008-5472.CAN-16-1346

Figure Lengend Snippet: (A) Scatter plot of RNA-seq analysis showing genes differentially expressed (q <0.001) in triplicate samples of HCT116 cells treated with a non-specific shRNA (NS, x-axis) or CNOT3 shRNA #3 (y-axis). Genes with (blue dots) and without (green or orange dots) nearby CNOT3 binding are colored and the top Gene Ontology terms (derived from Ingenuity analysis) enriched among genes affected by CNOT3 deficiency are shown. (B) Proportions of CNOT3 binding near genes that are differentially expressed (top) or unaffected (bottom) in CNOT3-depleted cells. (C) qRT-PCR verification of increased transcript levels of selected genes derepressed in CNOT3-deficient HCT116 cells. (D) Representative gene expression changes occurring in CNOT3-depleted HCT116 cells are rescued by shRNA-resistant cDNA, indicating shRNA specificity. (E) Heatmaps showing presence or absence of RNA polymerase II (Pol2), H3K4me3, DHS, DNA methylation and mRNA expression in parental HCT116 cells at CNOT3-bound promoters. Selected genes affected by CNOT3 depletion are listed. (F) Representative IGV traces for each class of CNOT3-bound promoters, showing various chromatin features and RNA-seq data from control and CNOT3-deficient cells.

Article Snippet: Immunodetection For immunohistochemistry, we used Ab against CNOT3 (Novus Biologicals, clone 4B8, 1:50), TRIM28 (Abnova, clone 4E6, 1:250), KI67 (DAKO, clone MIB-1, 1:50) or CTNNB1 (BD Transduction Laboratories, clone 14/Beta-Catenin, 1:250) diluted in Tris-buffered saline (TBS, pH 8.0) containing 1% bovine serum albumin.

Techniques: RNA Sequencing, shRNA, Binding Assay, Derivative Assay, Quantitative RT-PCR, Gene Expression, DNA Methylation Assay, Expressing, Control

Journal: Cancer research

Article Title: Transcriptional regulator CNOT3 defines an aggressive colorectal cancer subtype

doi: 10.1158/0008-5472.CAN-16-1346

Figure Lengend Snippet: (A) Gene Set Enrichment Analysis of CNOT3-regulated genes with respect to distinct components of the ESC program. The table expresses color-coded normalized enrichment scores (NES) and representative GSEA plots for modules increased (top), unaffected (center) or decreased (bottom) in expression in CNOT3-depleted HCT116 cells are shown. All listed NES values reflect significant (P <0.0001) enrichments. (B) Heatmaps of CNOT3-bound genes in the hypermethylated (top) and ESC (bottom) modules in HCT116 and Ca9, in relation to RAD21 (as a control) and MAX binding in HCT116 cells.

Article Snippet: Immunodetection For immunohistochemistry, we used Ab against CNOT3 (Novus Biologicals, clone 4B8, 1:50), TRIM28 (Abnova, clone 4E6, 1:250), KI67 (DAKO, clone MIB-1, 1:50) or CTNNB1 (BD Transduction Laboratories, clone 14/Beta-Catenin, 1:250) diluted in Tris-buffered saline (TBS, pH 8.0) containing 1% bovine serum albumin.

Techniques: Expressing, Control, Binding Assay

Journal: Cancer research

Article Title: Transcriptional regulator CNOT3 defines an aggressive colorectal cancer subtype

doi: 10.1158/0008-5472.CAN-16-1346

Figure Lengend Snippet: (A) IHC for CNOT3 in representative CRC specimens with typical (<1% to 5% of stained cells) or high (>5% to 20% of stained cells) CNOT3+ cell fractions. Scale bar, 50 µm. (B) Association of high CNOT3+ cell fractions with increasing CRC stage. (C) Kaplan-Meier survival analysis of patients with CRC stages II and III (combined, left, N=307) or stage II only (right, N=237), showing reduced long-term disease-free survival in cases with excess CNOT3. (D) Multivariate analysis in stages II and III (combined) or in stage II CRC of the influence of various factors, including high CNOT3+ cell fraction, on patient survival. (E) Kaplan-Meier survival analysis of patients whose CRCs did (red) or did not (blue) overexpress TRIM28. (F) Kaplan-Meier survival analysis of 296 patients with microsatellite-stable (MSI-negative) tumors with low or high CNOT3 mRNA levels in CRC stages II and III from The Cancer Genome Atlas collection.

Article Snippet: Immunodetection For immunohistochemistry, we used Ab against CNOT3 (Novus Biologicals, clone 4B8, 1:50), TRIM28 (Abnova, clone 4E6, 1:250), KI67 (DAKO, clone MIB-1, 1:50) or CTNNB1 (BD Transduction Laboratories, clone 14/Beta-Catenin, 1:250) diluted in Tris-buffered saline (TBS, pH 8.0) containing 1% bovine serum albumin.

Techniques: Staining

Journal: Acta Neuropathologica

Article Title: Isoglutaminyl cyclase contributes to CCL2-driven neuroinflammation in Alzheimer’s disease

doi: 10.1007/s00401-015-1395-2

Figure Lengend Snippet: Cocktails of antibodies used for double and triple labeling immunohistochemistry

Article Snippet: The goat anti-CCL2 antiserum sc-1784 (St. Cruz) and the mouse anti-CCL2 antibody (clone 4B8; Probiodrug, Halle/Saale, Germany) showed a distinct staining pattern for CCL2 around nuclei of mouse primary neurons as well as of neurons in mouse brain tissue.

Techniques: Labeling

Journal: Acta Neuropathologica

Article Title: Isoglutaminyl cyclase contributes to CCL2-driven neuroinflammation in Alzheimer’s disease

doi: 10.1007/s00401-015-1395-2

Figure Lengend Snippet: Neuronal isoQC expression, subcellular localization and co-expression of CCL2 in mouse brain and primary neurons. a IsoQC was strictly co-localized with the neuronal marker HuC/D in brain sections of 17-month-old wild-type mice demonstrating a neuron-specific expression. Cells which did not display HuC/D immunoreactivity were also negative for isoQC labeling ( arrows ). b IsoQC was co-localized with cellular compartment markers calreticulin and syntaxin-6, consistent with a subcellular localization in endoplasmic reticulum and Golgi apparatus. c The putative isoQC substrate CCL2 was found to be co-expressed by isoQC-immunoreactive neurons in cortex of wild-type mice ( top ) and in primary neuronal cultures ( bottom ). d The conversion of CCL2 by recombinant isoQC was analyzed in a kinetic assay. The progress curve ( black trace ) was in accordance with a curve modeled according to the integrated form of the Michaelis–Menten equation ( red trace ), enabling the determination of the kinetic parameters K M (19.8 ± 0.4 μM) and k cat (0.76 ± 0.01 s −1 )

Article Snippet: The goat anti-CCL2 antiserum sc-1784 (St. Cruz) and the mouse anti-CCL2 antibody (clone 4B8; Probiodrug, Halle/Saale, Germany) showed a distinct staining pattern for CCL2 around nuclei of mouse primary neurons as well as of neurons in mouse brain tissue.

Techniques: Expressing, Marker, Labeling, Recombinant, Kinetic Assay

Journal: Acta Neuropathologica

Article Title: Isoglutaminyl cyclase contributes to CCL2-driven neuroinflammation in Alzheimer’s disease

doi: 10.1007/s00401-015-1395-2

Figure Lengend Snippet: Regulation of isoQC and CCL2 expression in Tg2576 mice. a The neocortical isoQC and CCL2 mRNA levels were increased by 135 and 55 %, respectively, in 17-month-old APP transgenic Tg2576 mice ( black bars ) compared to wild-type littermates ( white bars ) as demonstrated by qRT-PCR analyses. Additionally, GFAP mRNA levels were increased by 115 %, indicating astrogliosis in Tg2576 mice. The isoQC and pGlu-CCL2 protein levels, however, were not affected in Tg2576 mice as measured by Western blot analysis and ELISA, respectively. b In 17-month-old wild-type (wt) and APP transgenic Tg2576 mice (tg) there was a predominantly neuronal expression of isoQC and CCL2 ( green immunofluorescence) as revealed by co-expression of HuC/D ( red immunofluorescence). The asterisks indicate the position of Abeta plaques in Tg2576 tissue. c In addition, aged Tg2576 mice—but not wild-type littermates—displayed astrocytic expression of isoQC and CCL2 in proximity of Abeta deposits following a gradient from the core towards the periphery of plaques ( I , within plaque core diameter; II , double plaque core diameter; III , triple plaque core diameter). d The astrocytic co-expression ( arrows ) of isoQC and CCL2 in proximity of Abeta plaques ( asterisks ) is visualized by the co-expression of the astrocyte marker GFAP ( red immunofluorescence). *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The goat anti-CCL2 antiserum sc-1784 (St. Cruz) and the mouse anti-CCL2 antibody (clone 4B8; Probiodrug, Halle/Saale, Germany) showed a distinct staining pattern for CCL2 around nuclei of mouse primary neurons as well as of neurons in mouse brain tissue.

Techniques: Expressing, Transgenic Assay, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Marker

Journal: Acta Neuropathologica

Article Title: Isoglutaminyl cyclase contributes to CCL2-driven neuroinflammation in Alzheimer’s disease

doi: 10.1007/s00401-015-1395-2

Figure Lengend Snippet: Co-regulation of isoQC and CCL2 in mouse primary astrocytes upon Abeta stimulation. a Immunocytochemical double labeling of isoQC ( green ) and CCL2 ( red ) with nuclear Hoechst counterstaining ( blue ) under control conditions and after stimulation with Abeta (5 μM) or pGlu-Abeta (5 μM) as indicated. Note the robust increase in the immunocytochemical labeling intensity for both proteins at 24 and 48 h and the decline at 72 h. b Quantification of immunocytochemical labeling revealed a highly significant time- and Abeta peptide-specific increase in isoQC and CCL2 immunoreactivity. Correlation analyses between isoQC and CCL2 immunocytochemical labeling demonstrated highly significant correlations in the expression of enzyme and substrate under control conditions ( b1 ), after stimulation with Abeta ( b2 ) and with pGlu-Abeta ( b3 ). c Quantification of isoQC and CCL2 mRNA expression under control conditions and after stimulation with Abeta and pGlu-Abeta for different periods of time as indicated ( N = 6 per time point). Note the absence of an increase of isoQC or CCL2 mRNA transcripts independent of the type and duration of the treatment. Correlation analyses of CCL2 mRNA levels plotted versus isoQC mRNA levels in individual astrocyte culture wells under control conditions and after (pGlu)-Abeta stimulation including all time points analyzed. Note the absence of a correlation between enzyme and substrate mRNA expression under control ( c1 ) and treatment conditions ( c2 , c3 )

Article Snippet: The goat anti-CCL2 antiserum sc-1784 (St. Cruz) and the mouse anti-CCL2 antibody (clone 4B8; Probiodrug, Halle/Saale, Germany) showed a distinct staining pattern for CCL2 around nuclei of mouse primary neurons as well as of neurons in mouse brain tissue.

Techniques: Labeling, Control, Expressing

Journal: Acta Neuropathologica

Article Title: Isoglutaminyl cyclase contributes to CCL2-driven neuroinflammation in Alzheimer’s disease

doi: 10.1007/s00401-015-1395-2

Figure Lengend Snippet: Immunohistological alterations of isoQC and CCL2 expression in AD cortex. a Immunohistochemistry for isoQC in human temporal cortex revealed a weak neuronal expression in control subjects. In corresponding tissue sections of AD subjects, an increased neuronal labeling intensity, in particular in layer III pyramidal neurons was observed. b A similar up-regulation in pyramidal layer III neurons was observed for CCL2. Additionally, both isoQC and CCL2 were induced in glia-like cells in proximity of Abeta plaques ( insets in a and b ). c Triple immunofluorescent labelings of isoQC with CCL2 and GFAP identified these glial cells as astrocytes surrounding Abeta deposits. d The astrocytic expression of isoQC and CCL2 in proximity of Abeta deposits followed a gradient from the core to the periphery of plaques ( I , within plaque core diameter; II , double plaque core diameter; III , triple plaque core diameter). **p < 0.01, ***p < 0.001

Article Snippet: The goat anti-CCL2 antiserum sc-1784 (St. Cruz) and the mouse anti-CCL2 antibody (clone 4B8; Probiodrug, Halle/Saale, Germany) showed a distinct staining pattern for CCL2 around nuclei of mouse primary neurons as well as of neurons in mouse brain tissue.

Techniques: Expressing, Immunohistochemistry, Control, Labeling

Journal: Acta Neuropathologica

Article Title: Isoglutaminyl cyclase contributes to CCL2-driven neuroinflammation in Alzheimer’s disease

doi: 10.1007/s00401-015-1395-2

Figure Lengend Snippet: Characteristics of isoQC, CCL2 and pGlu-Abeta accumulation in AD and correlation analyses with MMSE. a Quantitative analyses revealed statistically significant increases of isoQC and CCL2 mRNA levels as well as pGlu-CCL2 protein in temporal cortex samples from AD cases compared to control subjects ( *p < 0.05). b There was a statistically significant correlation between higher isoQC mRNA levels and the decline in MMSE ( r = −0.7220; p = 0.0080). c Likewise, a statistically significant correlation between higher CCL2 mRNA levels and the decline in MMSE ( r = −0.6124; p = 0.0343) was detected. d There was no correlation between isoQC and CCL2 transcript levels in the human brain samples analyzed. e A strong positive correlation between CCL2 mRNA and protein levels was detected ( r = 0.8552; p = 0.0004). f As for CCL2 mRNA, an inverse correlation between pGlu-CCL2 protein and MMSE was revealed ( r = −0.6336; p = 0.0270). While there was no correlation between isoQC mRNA levels and total Abeta42 peptide concentrations ( g ), a significant correlation between isoQC transcript levels and pGlu-Abeta concentrations was established ( r = 0.6663; p = 0.0180) ( h )

Article Snippet: The goat anti-CCL2 antiserum sc-1784 (St. Cruz) and the mouse anti-CCL2 antibody (clone 4B8; Probiodrug, Halle/Saale, Germany) showed a distinct staining pattern for CCL2 around nuclei of mouse primary neurons as well as of neurons in mouse brain tissue.

Techniques: Control