p hp1γ  (Cell Signaling Technology Inc)

Journal: Epigenetics & Chromatin

Article Title: Functional impact of Aurora A-mediated phosphorylation of HP1γ at serine 83 during cell cycle progression

doi: 10.1186/1756-8935-6-21

Figure Lengend Snippet: Biphasic P-Ser 83 - HP1 γ is observed during cell cycle progression . (A,B,C) P-Ser 83 -HP1γ levels vary during the cell cycle. Panoramic view of a growing population of HeLa cells staining with anti-P-Ser 83 -HP1γ ( A , green) demonstrates that the signal for this protein varies in intensity in different cells. Cells were counterstained with DAPI ( B , blue) to show DNA and overlay is shown in (C) . Three main populations are observed according to the strength of the signal, namely strong, moderate and negligible. Scale bar represents 20 μM. (D,E,F) P-Ser 83 -HP1γ displays punctate euchromatic localization in G 1 phase. Localization of P-Ser 83 -HP1γ ( D , green) was determined in cyclin D-positive cells ( E , red), indicative of G 1 phase, as shown with arrows and in overlay (F) . (G,H,I) Levels of P-Ser 83 -HP1γ diminish during S phase. Negligible P-Ser 83 -HP1γ signal ( G , green) is found in the majority of cells undergoing S phase (arrows), as determined by EdU positively labeled cells ( H , red). Overlay is shown in (I) . (J,K,L) P-Ser 83 -HP1γ levels increase upon G 2 entry. Cyclin B-positive cells ( K , red), before nuclear envelope breakdown (G 2 ), not only shows the P-Ser 83 -HP1γ signal ( J , green) as a strong punctate pattern in euchromatin, but also with separating centrosomes ( L , overlay). Scale bar represents 10 μM for panels ( D to L ). (M,N,O,P,Q,R) P-Ser 83 -HP1γ levels persist through mitosis. Cyclin B-positive, prometaphase cell demonstrates an increase in P-Ser 83 -HP1γ in association with separating centrosomes (M) . Metaphase cell shows the P-Ser 83 -HP1γ remains localized to centrosomes, which are forming the mitotic spindle (N) . Early (O) and late (P) anaphase, as well as telophase (Q) cells are shown, where the P-Ser 83 -HP1γ signal intensity at the centrosomes is decreased as cells prepare to complete cell division. P-Ser 83 -HP1γ signal within euchromatic regions is again observed during cytokinesis (R) . Scale bar represents 5 μM for panels (M to R). DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2´-deoxyuridine; P-Ser 83 -HP1γ, phosphorylation of HP1γ at serine 83.

Article Snippet: The primary antibodies were used at the following dilutions: P-Ser 83 -HP1γ, 1:200; and γ-tubulin, 1:500 (Sigma-Aldrich); Aurora A, 1:50; and Aurora B, 1:50 (BD Biosciences Pharmingen); cyclin B1, 1:500; cyclin B2, 1:100; and CDK1, 1:40 (Abcam); and cyclin D3, 1:200 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Staining, Labeling

Journal: Epigenetics & Chromatin

Article Title: Functional impact of Aurora A-mediated phosphorylation of HP1γ at serine 83 during cell cycle progression

doi: 10.1186/1756-8935-6-21

Figure Lengend Snippet: Levels of P - Ser 83 - HP1γ are cell cycle - dependent , increasing significantly in G 2 / M . (A) Inhibition of HP1γ phosphorylation in vivo by the cell cycle inhibitor, roscovitine. HeLa cells incubated with roscovitine, an inhibitor of cell cycle progression at the G 1 /S and G 2 /M checkpoints, display a dose-dependent inhibition of phosphorylation as shown by anti-P-Ser 83 -HP1γ (top). α-tubulin is shown as a loading control (bottom). (B) P-Ser 83 -HP1γ levels are high in mitotic arrested cells. Cell extracts were obtained from a normal cycling population (con), cells treated with aphidicolin (aph) to arrest cells in G 1 /S phase (G 1 /S), or mitotic-arrested cells (G 2 /M) from treatment with nocodazole (noc). An increase of P-Ser 83 -HP1γ levels in mitosis is shown by comparison of anti-P-Ser 83 -HP1γ (top) with total HP1γ (bottom). (C) P-Ser 83 -HP1γ levels through the cell cycle. HeLa cells were synchronized by double thymidine block and cell extracts were obtained at subsequent time points of release. P-Ser 83 -HP1γ levels are highest approximately 8 to 10 hours post-release, which corresponds to an increase in the presence of other mitotic markers, including P-Ser 10 -H3, Aurora A and Aurora B, indicating M phase entry. The relative intensity indicated below was calculated as P-Ser 83 -HP1γ/pan-HP1γ ratios and normalization with the ratio of 0 hour. aph, aphidicolin; con, control; noc, nocodazole; P-Ser 10 -H3, phosphorylation of histone H3 at serine 10; P-Ser 83 -HP1γ, phosphorylation of HP1γ at serine 83.

Article Snippet: The primary antibodies were used at the following dilutions: P-Ser 83 -HP1γ, 1:200; and γ-tubulin, 1:500 (Sigma-Aldrich); Aurora A, 1:50; and Aurora B, 1:50 (BD Biosciences Pharmingen); cyclin B1, 1:500; cyclin B2, 1:100; and CDK1, 1:40 (Abcam); and cyclin D3, 1:200 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Inhibition, In Vivo, Incubation, Blocking Assay

Journal: Epigenetics & Chromatin

Article Title: Functional impact of Aurora A-mediated phosphorylation of HP1γ at serine 83 during cell cycle progression

doi: 10.1186/1756-8935-6-21

Figure Lengend Snippet: P - Ser 83 - HP1γ colocalizes with Aurora A at the mitotic spindle . Representative images are shown for localization in mitotic HeLa cells. (A,B,C) Colocalization of P-Ser 83 -HP1γ ( A , green) is shown with Aurora A ( B , red) at the spindle poles. The overlay is shown in (C) . (D,E,F) Cells in metaphase were also stained for P-Ser 83 -HP1γ ( D , green) and Aurora B ( E , red), which demonstrates that there is no colocalization of these two proteins as observed in the overlay (F) . (G,H,I,J,K,L) P-Ser 83 -HP1γ ( G , J , green) was confirmed to be present at the spindle poles through co-staining with γ-tubulin ( H , red) as well as α-tubulin ( K , red) as shown in the overlays ( I , L ). (M,N,O,P,Q,R,S,T,U) In addition, CDK1 ( N , red), cyclin B1 ( Q , red) and cyclin B2 ( T , red) were each shown to co-localize with P-Ser 83 -HP1γ ( M , P , S , green) as shown by overlays ( O , R , U ). Cells were counterstained with DAPI (blue) to show DNA. Scale bar represents 5 μM. CDK1, cyclin-dependent kinase 1; DAPI, 4',6-diamidino-2-phenylindole; P-Ser 83 -HP1γ, phosphorylation of HP1γ at serine 83.

Article Snippet: The primary antibodies were used at the following dilutions: P-Ser 83 -HP1γ, 1:200; and γ-tubulin, 1:500 (Sigma-Aldrich); Aurora A, 1:50; and Aurora B, 1:50 (BD Biosciences Pharmingen); cyclin B1, 1:500; cyclin B2, 1:100; and CDK1, 1:40 (Abcam); and cyclin D3, 1:200 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Staining

Journal: Epigenetics & Chromatin

Article Title: Functional impact of Aurora A-mediated phosphorylation of HP1γ at serine 83 during cell cycle progression

doi: 10.1186/1756-8935-6-21

Figure Lengend Snippet: Aurora A phosphorylates Ser 83 - HP1γ in G 2 / M . (A) Aurora kinases phosphorylate Ser 83 in vitro. In vitro kinase assays were performed on GST fusion proteins, which demonstrate that wild type, not S83A-HP1γ mutant, is phosphorylated by Aurora kinases. (B) Aurora A siRNA reduces P-Ser 83 -HP1γ. Aurora A siRNA significantly reduced P-Ser 83 -HP1γ, whereas Aurora B siRNA only slightly reduced P-Ser 83 -HP1γ (top). Aurora A ( AURKA ) and Aurora B ( AURKB ) were effectively knocked-down (middle panels). Relative intensities were calculated as P-Ser 83 -HP1γ/β-actin ratios. (C) Wild type Aurora kinases increase P-Ser 83 -HP1γ. CHO cells, with low basal P-Ser 83 -HP1γ, demonstrated increased P-Ser 83 -HP1γ (top) upon transfection of Aurora kinases (Myc-tag; middle). (D) Aurora A-dominant negative (DN) reduces P-Ser 83 -HP1γ. P-Ser 83 -HP1γ (top) was significantly reduced with Aurora A-DN in BxPC3, epithelial cells with high basal P-Ser 83 -HP1γ. Aurora B-DN also reduced P-Ser 83 -HP1γ, although still detected. Aurora-DN levels are shown by Myc-tag. β-actin serves as loading control (B, C, D; bottom). (E,F) Aurora A-DN abolishes mitotic P-Ser 83 -HP1γ. Representative images of overlays with DAPI counterstain are shown for P-Ser 83 -HP1γ (green) with control (E) or Aurora A-DN (F). Typical P-Ser 83 -HP1γ localization was still observed in interphase with Aurora A-DN, but disrupted in metaphase (arrows). Scale bar represents 10 μM. (G,H) . Pharmacological inhibition of Aurora A, but not Aurora B, inhibits P-Ser 83 -HP1γ. Aurora A inhibition with MLN8237 was confirmed by loss of activated P-Thr 288 relative to total Aurora A (G, lower panels). P-Ser 83 -HP1γ was significantly reduced with MLN8237, without affecting pan-HP1γ (G, upper panels). Conversely, Aurora B inhibition by hesperidin did not reduce P-Ser 83 -HP1γ (H, top). Aurora B inhibition was confirmed by P-Ser 10 -H3, a well-known Aurora B target (H, bottom). CHO, Chinese hamster ovary; DAPI, 4',6-diamidino-2-phenylindole; DN, dominant negative; GST, glutathione S-transferase; P-Ser 10 -H3, phosphorylation of histone H3 at serine 10; P-Ser 83 -HP1γ, phosphorylation of HP1γ at serine 83; P-Thr 288 , phosphorylation of Aurora A at threonine 288; Ser 83 , serine 83.

Article Snippet: The primary antibodies were used at the following dilutions: P-Ser 83 -HP1γ, 1:200; and γ-tubulin, 1:500 (Sigma-Aldrich); Aurora A, 1:50; and Aurora B, 1:50 (BD Biosciences Pharmingen); cyclin B1, 1:500; cyclin B2, 1:100; and CDK1, 1:40 (Abcam); and cyclin D3, 1:200 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: In Vitro, Mutagenesis, Transfection, Dominant Negative Mutation, Inhibition

Journal: Epigenetics & Chromatin

Article Title: Functional impact of Aurora A-mediated phosphorylation of HP1γ at serine 83 during cell cycle progression

doi: 10.1186/1756-8935-6-21

Figure Lengend Snippet: P-Ser 83 - HP1γ is necessary for proper mitotic function . (A) Stable knockdown of HP1γ in HeLa cells. Western blot of HP1γ levels (top) is shown from HeLa cell lysates to confirm stable lentiviral-mediated shHP1γ compared to shCTRL. α-tubulin serves as a loading control (bottom). (B) HP1γ knockdown eliminates P-Ser 83 -HP1γ at the spindle poles. Representative images are shown for immunofluorescence on shCTRL and shHP1γ HeLa cells to demonstrate specific loss of P-Ser 83 -HP1γ (green) staining. Co-staining with γ-tubulin (red) was performed to establish the localization of the spindle poles. Cells were counterstained with DAPI and the overlay is shown. Scale bar represents 5 μM. ( C ) Mitotic aberrations caused by HP1γ knockdown are rescued by wild type, but not S83A-HP1γ mutant. Mitotic aberrations were quantified for shCTRL and shHP1γ cells. In order to determine if Ser 83 phosphorylation plays a role in this function, shHP1γ cells were infected with adenovirus carrying wild type or S83A-HP1γ mutant. While reintroduction of wild type HP1γ was able to significantly rescue this effect, S83A-HP1γ mutant was not, implicating Aurora A-mediated phosphorylation in this phenomenon. For each condition, 200 mitotic cells were analyzed. Western blot is shown of endogenous HP1γ levels (inlay, top) as well as transduced His-tagged wild type and S83A-HP1γ mutant proteins (arrow). α-tubulin serves as a loading control (inlay, bottom). *Transduction with EV control did not change the number of abnormalities observed with shHP1γ. (D) Mitotic aberrations observed in stable shHP1γ cells include multipolar spindles, centrosome disruption and lagging, unorganized chromosomes. Representative images are shown for the types of observed mitotic aberrations. γ-tubulin (red) marks spindle poles with DAPI counterstain to show condensed mitotic chromosomes. Scale bar represents 5 μM. DAPI, 4',6-diamidino-2-phenylindole; EV, empty vector; P-Ser 83 -HP1γ, phosphorylation of HP1γ at serine 83; Ser 83 , serine 83; shCTRL, shRNA control; shHP1γ, shRNA knockdown of HP1γ; shRNA, short hairpin RNA.

Article Snippet: The primary antibodies were used at the following dilutions: P-Ser 83 -HP1γ, 1:200; and γ-tubulin, 1:500 (Sigma-Aldrich); Aurora A, 1:50; and Aurora B, 1:50 (BD Biosciences Pharmingen); cyclin B1, 1:500; cyclin B2, 1:100; and CDK1, 1:40 (Abcam); and cyclin D3, 1:200 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Western Blot, Immunofluorescence, Staining, Mutagenesis, Infection, Transduction, Plasmid Preparation, shRNA

Journal: Epigenetics & Chromatin

Article Title: Functional impact of Aurora A-mediated phosphorylation of HP1γ at serine 83 during cell cycle progression

doi: 10.1186/1756-8935-6-21

Figure Lengend Snippet: P-Ser 83 - HP1γ status alters cell proliferation and cell cycle - related gene networks . (A) P-Ser 83 -HP1γ plays a role in cell proliferation. Cell proliferation was measured in the presence of control (EV), wild type HP1γ, the nonphosphorylatable (S83A)- or phosphomimetic (S83D)-HP1γ mutants by EdU incorporation, using both FACS and microscopy. Wild type HP1γ demonstrated only a slight increase in EdU incorporation compared to EV. However, while mutation of S83A-HP1γ decreased the levels of EdU, the S83D-HP1γ mutant had a significant increase in levels of EdU incorporation over control cells. Western blot controlling expression of His-tagged wild type and mutant HP1γ proteins is shown (top, inlay). A representative immunofluorescence image (40 × magnification) of EdU-positive cells (green) is shown below each respective experimental condition. Cells were counterstained with DAPI to detect total number of cells present in a field. * P values <0.05. (B) Genome-wide expression analysis of HP1γ highlights consequences of Ser 83 phosphorylation. Hierarchical clustering of significant targets ( P value <0.05) from Affymetrix Human Gene 1.0 ST microarray demonstrates the close relationship between EV and the nonphosphorylatable S83A-HP1γ mutant. Large clusters of genes show deregulation in the presence of either the nonphosphorylatable (S83A)- or phosphomimetic (S83D)-HP1γ mutants. (C) P-Ser 83 -HP1γ status influences the expression of G 2 /M-related genes. Gene Ontology (GO) ANOVA reveals significant differential expression of genes by both wild type and mutant HP1γ in functional groupings related to mitosis and cell division, again indicating that the presence of an active phosphorylation site at Ser 83 is necessary for proper mitotic function as a sizeable number of targets are deregulated in the presence of the HP1γ mutants with altered phosphorylation abilities. ANOVA, analysis of variance; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2´-deoxyuridine; EV, empty vector; FACS, fluorescence-activated cell sorting; GO, Gene Ontology; P-Ser 83 -HP1γ, phosphorylation of HP1γ at serine 83; Ser 83 , serine 83.

Article Snippet: The primary antibodies were used at the following dilutions: P-Ser 83 -HP1γ, 1:200; and γ-tubulin, 1:500 (Sigma-Aldrich); Aurora A, 1:50; and Aurora B, 1:50 (BD Biosciences Pharmingen); cyclin B1, 1:500; cyclin B2, 1:100; and CDK1, 1:40 (Abcam); and cyclin D3, 1:200 (Cell Signaling Technology, Danvers, MA, USA).

Techniques: Microscopy, Mutagenesis, Western Blot, Expressing, Immunofluorescence, Genome Wide, Microarray, Functional Assay, Plasmid Preparation, Fluorescence, FACS

Journal: The Journal of Clinical Investigation

Article Title: CDCP1 overexpression drives prostate cancer progression and can be targeted in vivo

doi: 10.1172/JCI131133

Figure Lengend Snippet: (A) Representative images of p-HP1γ. Senescence associated-β-galactosidase (SA-β-Gal) and Cyclin D1 staining in the anterior prostates of WT, CDCP1, Ptenpc–/–, and CDCP1 Ptenpc–/– mice. Scale bars: 125 μm. (B) Western blot analysis of p21, Cyclin D1, COUP-TFII, Smad4, and p53 in anterior prostate glands from the indicated genotypes. (C) qRT-PCR analysis of c-Myc, Cyclin D1, COUP-TFII, p21, p27, and p16 expression in prostates from 12- to 16-week-old Ptenpc–/– and CDCP1 Ptenpc–/– mice (n = 3). (D) Western blot analysis of Pten–/– and CDCP1 Pten–/– MEFs treated with saracatinib (100 nM) for 12 hours. (E) Representative images of SA-β-Gal staining in Pte–/– and CDCP1 Pten–/– MEFs treated with saracatinib (100 nM) and DMSO for 12 hours. Scale bars: 125 μm. Bar graph shows the fold change in growth by crystal violet in Pten–/– and CDCP1 Pten–/– MEFs treated with saracatinib (100 nM) or DMSO as control (n = 3). (F) Western blot analysis of Pten–/– and CDCP1 Pten–/– MEFs transfected with si-c-Myc and control si-scramble (si-Ctrl) after 48 hours. (G) Representative images of SA-β-Gal staining in Pten–/– and CDCP1 Pten–/– MEFs transfected with si-c-Myc and si-Ctrl after 48 hours. Scale bars: 125 μm. Bar graph shows the fold change in growth by crystal violet in Pten–/– and CDCP1 Pten–/– MEFs transfected with si-c-Myc and si-Ctrl (n = 3). (H) Schemes of Cyclin D1 and COUP-TFII promoters. qRT-PCR of ChIP-analysis showing the binding of c-Myc to COUP-TFII promoter and c-Myc and Smad4 to Cyclin D1 promoters in Pten–/– and CDCP1 Pten–/– MEFs. Normal mouse IgG serves as negative control (n = 2). Error bars indicate SD. *P < 0.05; **P < 0.01. Statistical test: 2-tailed t test.

Article Snippet: For IHC the following antibodies were used: Ki-67 (Thermo Fisher Scientific, clone SP6, catalog RM-9106-R7; rabbit polyclonal; unmasked water bath 98°C, pH 6, 20 minutes; Lab Vision dilution ready to use); CDCP1 (Cell Signaling Technology, catalog 4115, rabbit polyclonal; unmasked water bath 98°C, pH 6, 20 minutes; 1:50); p-HP1γ-Ser83 (Cell Signaling Technology, catalog 2600, unmasked water bath 98°C, pH 6, 20 minutes; 1:50); Cyclin D1 (Cell Signaling Technology, catalog 2978S); AR (N-20) (Santa Cruz Biotechnology, catalog SC-816, rabbit polyclonal; unmasked water bath 98°C, pH 6, 20 minutes; 1:300); wide spectrum cytokeratin (pankeratin) (DAKO, catalog Z0622; rabbit polyclonal; unmasked water bath 98°C, pH 9, 20 minutes; 1:2000).

Techniques: Staining, Western Blot, Quantitative RT-PCR, Expressing, Transfection, Binding Assay, Negative Control