Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Chromobox Protein Homolog 3 Is Essential for Stem Cell Differentiation to Smooth Muscles In Vitro and in Embryonic Arteriogenesis

doi: 10.1161/atvbaha.111.230110

Figure Lengend Snippet: Figure 1. Cbx3 is upregulated during SMC differentiation. A, Induction of SMC differentiation markers during stem cell differ- entiation. Undifferentiated ES cells were plated onto dishes coated with 5 g/mL collagen IV and cultured in DM. Total RNA from undifferentiated ES cells (d0) or differentiating ES cells at days 3 (d3), 5 (d5), and 7 (d7) were harvested and subjected to real-time PCR analysis with specific primer pairs for SMA and SM-MHC. B, mRNA induction of Cbx3 is upregulated during SMC differentiation. C, Nuclear protein from undifferentiated ES cells (d0) or differentiating ES cells at the indicated time points was harvested and subjected to Western blot analysis. KD indi- cates kilodaltons. D, Nuclear localization of Cbx3 in differentiat- ing ES cells. Immunofluorescence staining was conducted on day 7 differentiating ES cells with antibodies against Cbx3. DAPI indicates 4,6-diamidino-2-phenylindole. The data presented here are representative or an average of 3 independent experi- ments. *P0.05 compared with control.

Article Snippet: The Cbx3 (or HP1) siRNA (sc-35590), Dia-1 siRNA (sc-35191), and fluorescein conjugated non-targeting control siRNA-C (sc-44240) were purchased from Santa Cruz Biotech.

Techniques: Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Staining, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Chromobox Protein Homolog 3 Is Essential for Stem Cell Differentiation to Smooth Muscles In Vitro and in Embryonic Arteriogenesis

doi: 10.1161/atvbaha.111.230110

Figure Lengend Snippet: Figure 2. Cbx3 is crucial for SMC differentiation from ES cells. A and B, Cbx3 knockdown downregulated SMC differentiation gene expression. Cbx3-specific siRNA and random siRNA con- trol were transfected into day 3 differentiating ES cells; after an additional 48 or 72 hours of culture, total RNA and protein were harvested and subjected to quantitative real-time RT-PCR (A) and Western blot analysis (B), respectively. KD indicates kilodal- tons. C and D, Enforced Cbx3 expression promotes SMC differ- entiation. Undifferentiated ES cells were nucleofected by nucleo- fector II with different amounts of Cbx3 expression plasmid pCMV5-Cbx3. Nucleofected cells were plated in dishes coated with 5 g/mL collagen IV and cultured for 3 to 4 days in DM. Total RNA and protein were harvested and subjected to real- time PCR analysis for gene expression (C) and Western blot analysis for protein levels (D), respectively. Appropriate amounts of empty vector pCMV5 were included as plasmid amount com- pensation. -Tubulin was included as internal control. The data presented here are representative or an average of 3 indepen- dent experiments. E to G, Flow cytometry analysis of the SMA expression pattern in the differentiated SMCs. Undifferentiated ES cells were nucleofected using nucleofector II with control (pCMV5) or Cbx3 expression plasmid pCMV5-Cbx3 (1 g/106

Article Snippet: The Cbx3 (or HP1) siRNA (sc-35590), Dia-1 siRNA (sc-35191), and fluorescein conjugated non-targeting control siRNA-C (sc-44240) were purchased from Santa Cruz Biotech.

Techniques: Knockdown, Gene Expression, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Plasmid Preparation, Cell Culture, Real-time Polymerase Chain Reaction, Control, Flow Cytometry

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Chromobox Protein Homolog 3 Is Essential for Stem Cell Differentiation to Smooth Muscles In Vitro and in Embryonic Arteriogenesis

doi: 10.1161/atvbaha.111.230110

Figure Lengend Snippet: Figure 3. The CD and CSD but not the Hinge component of Cbx3 are responsible for Cbx3-induced SMC differentiation. A, Schematic illustration of Cbx3 gene structure, Cbx3 domain deletion, and PxVxL motif potential binding site mutation. CD indicates CD deletion; Hinge, Hinge domain deletion; CSD, CSD deletion; 165/8, aa 165 to 168 mutation; KD, kilodaltons. B and C, The CD and CSD but not Hinge of Cbx3 were respon- sible for its potential to promote SMC differentiation. Undifferen- tiated ES cells were nucleofected using nucleofector II with full- length Cbx3 pCMV5-HA-Cbx3 or truncated/mutated forms of Cbx3 expression plasmids (CD, Hinge, CSD, and 165/8) (1 g per 106 cells). Nucleofected cells were plated in dishes coated with 5 g/mL collagen IV and cultured for 3 to 4 days in DM. Total RNA and protein were harvested and subjected to real-time PCR analysis for gene expression (B) and Western blot analysis for protein levels (C), respectively. -Tubulin was included as internal control. D, Bar graphs represent meanSEM of densitometric analysis (n3) of the relative pro- tein levels of SMA and SM-MHC. *P0.05 (full-length vs pCMV5), #P0.05 (truncated/mutated forms of Cbx3 vs full- length Cbx3).

Article Snippet: The Cbx3 (or HP1) siRNA (sc-35590), Dia-1 siRNA (sc-35191), and fluorescein conjugated non-targeting control siRNA-C (sc-44240) were purchased from Santa Cruz Biotech.

Techniques: Binding Assay, Mutagenesis, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Gene Expression, Western Blot, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Chromobox Protein Homolog 3 Is Essential for Stem Cell Differentiation to Smooth Muscles In Vitro and in Embryonic Arteriogenesis

doi: 10.1161/atvbaha.111.230110

Figure Lengend Snippet: Figure 4. Cbx3 mediates SMC differentiation gene expression through interaction with Dia-1. A, Dia-1 knockdown abolished Cbx3-induced SMC differentiation. Differentiating ES cells were cotransfected with pCMV5- or pCMV5-Cbx3 and random con- trol siRNA or Dia-1-specific siRNA. Total RNA and protein were harvested and subjected to real-time PCR analysis (left) and Western blot analysis (right), respectively. -Tubulin was included as internal control. *P0.05 (control siRNA/pCMV5- Cbx3 vs control siRNA/pCMV5), &P0.05 (Dia-1 siRNA/pCMV5- Cbx3 vs control siRNA/pCMV5-Cbx3). KD indicates kilodaltons. B, Dia-1 protein levels were not changed by Cbx3 overexpres- sion. C, One PxVxL motif within the Dia-1 protein. D, Coimmu- noprecipitation assay showed the interactions of Cbx3/Dia-1/ SRF. The data presented in A, B, and D are representative or an average of 3 independent experiments. E, Coimmunoprecipita- tion assay revealed that aa 165 to 168 of Cbx3 were responsi- ble for the interaction between Cbx3 and Dia-1. Left, represen- tative data. Right, meanSEM of densitometric analysis (n3), *P0.05 (CSD or 165/8 vs full-length). Relative level of Dia-1 binding was defined as the ratio of the densitometric value of Dia-1 bands in the HA-IP lanes vs that of the related input lanes, with that of the full length set as 1.0. IP indicates immu- noprecipitation; IB, immunoblot; HA-IP, IP with anti- hemagglutinin in antibody.

Article Snippet: The Cbx3 (or HP1) siRNA (sc-35590), Dia-1 siRNA (sc-35191), and fluorescein conjugated non-targeting control siRNA-C (sc-44240) were purchased from Santa Cruz Biotech.

Techniques: Gene Expression, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Control, Binding Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Chromobox Protein Homolog 3 Is Essential for Stem Cell Differentiation to Smooth Muscles In Vitro and in Embryonic Arteriogenesis

doi: 10.1161/atvbaha.111.230110

Figure Lengend Snippet: Figure 5. Cbx3 promotes Dia-1 nuclear translocalization and SRF recruitment to the promoter of SMC specific genes. A and B, Over- expression of full-length Cbx3 (pCbx3-FL-HA) but not CSD-deleted Cbx3 (pCbx3-CSD-HA) promoted Dia-1 nuclear translocalization. ES cells were nucleofected using nucleofector II with control plasmids (pCMV5), pCbx3-FL-HA, or pCbx3-CSD-HA (1 g per 106

Article Snippet: The Cbx3 (or HP1) siRNA (sc-35590), Dia-1 siRNA (sc-35191), and fluorescein conjugated non-targeting control siRNA-C (sc-44240) were purchased from Santa Cruz Biotech.

Techniques: Over Expression, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Chromobox Protein Homolog 3 Is Essential for Stem Cell Differentiation to Smooth Muscles In Vitro and in Embryonic Arteriogenesis

doi: 10.1161/atvbaha.111.230110

Figure Lengend Snippet: Figure 6. Disruption of Cbx3 in NCCs results in the maldevelopment of 3rd and 4th of branchial arch artery and death of chick embryos. A, Embryos were electroporated with pCAb-Cbx3 DNF-GFP or pCAb-GFP and observed on the 2nd and 3rd days under a dissection micro- scope. In the right panels, note the abnormal structure of the branchial arch artery (black arrows in green boxes) and at the hemorrhage site (green arrow). Green arrow in the bottom right panel indicates the broken 4th branchial arch artery, which was sectioned and stained by hematoxylin/eosin (inset). Scale bars1 mm. B, 2 analysis was performed using SPSS 17.0 software on the both groups, and pCAb-Cbx3 DNF was found to significantly affect chick embryonic development (P0.00001). C, Defect in branchial arch artery caused by Cbx3 misex- pression in chick embryos. Ca and Cb, Representative chick embryos electroporated with pCAb-Cbx3 DNF-GFP (Ca) or pCAb-GFP (Cb). Images were taken before harvest. Notably, embryos from pCAb-Cbx3 DNF-GFP group showed invisible blood circulation of branchial arch

Article Snippet: The Cbx3 (or HP1) siRNA (sc-35590), Dia-1 siRNA (sc-35191), and fluorescein conjugated non-targeting control siRNA-C (sc-44240) were purchased from Santa Cruz Biotech.

Techniques: Disruption, Dissection, Staining, Software

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: For stable shRNA-mediated HP1γ knockdown, control or HP1γ-specific shRNA lentiviral particles (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) were used to infect cells according to the manufacturer’s protocol, followed by puromycin selection (2 μg/ml).

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: For stable shRNA-mediated HP1γ knockdown, control or HP1γ-specific shRNA lentiviral particles (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) were used to infect cells according to the manufacturer’s protocol, followed by puromycin selection (2 μg/ml).

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: For stable shRNA-mediated HP1γ knockdown, control or HP1γ-specific shRNA lentiviral particles (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) were used to infect cells according to the manufacturer’s protocol, followed by puromycin selection (2 μg/ml).

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: For stable shRNA-mediated HP1γ knockdown, control or HP1γ-specific shRNA lentiviral particles (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) were used to infect cells according to the manufacturer’s protocol, followed by puromycin selection (2 μg/ml).

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: For stable shRNA-mediated HP1γ knockdown, control or HP1γ-specific shRNA lentiviral particles (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) were used to infect cells according to the manufacturer’s protocol, followed by puromycin selection (2 μg/ml).

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: For stable shRNA-mediated HP1γ knockdown, control or HP1γ-specific shRNA lentiviral particles (Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA) were used to infect cells according to the manufacturer’s protocol, followed by puromycin selection (2 μg/ml).

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

Journal: Nutrients

Article Title: Anti-Stress, Glial- and Neuro-Differentiation Potential of Resveratrol: Characterization by Cellular, Biochemical and Imaging Assays

doi: 10.3390/nu12030671

Figure Lengend Snippet: Molecular changes in control and resveratrol-treated stressed cells. ( A ). Western blot showing induction of DNA damage signaling and growth arrest with various chemical stresses listed in ( B ). Resveratrol-treated cells showed considerable reversal of the molecular changes. ( C ). Immunostaining showing induction of DNA damage (γH2AX) and senescence (HP1γ) in stressed cells. Resveratrol-treated cells showed considerable recovery. The decrease in mortalin in cells stressed with some stresses was recovered with resveratrol treatment.

Article Snippet: Primary antibodies against Luciferase (AbCam, ab16466), γH2AX (Cell Signaling, 9718S), pCHK1 (Cell Signaling, 2344S), pCHK2 (Cell Signaling, 2197P), HP1γ (Merck Millipore, 05–690), Mortalin [ ], β-actin (AbCam, ab49900), GFAP (Sigma-Aldrich, G9269), Nestin (Santa Cruz, SC-23927), MAP2 (Sigma-Aldrich, M3696), PSD-95 (Santa Cruz, SC-32290), Neuropsin (Santa Cruz, SC-134600), GAP43 (Santa Cruz, SC-33705), NCAM (Santa Cruz, SC-10735), Survivin (Santa Cruz, SC-10811), Vimentin (Santa Cruz, SC-6260), NeuN (AbCam, ab177487), TCF4 (Santa Cruz, SC-166699), SMAD2/3 (Cell Signaling, 8685S), pSMAD2/3 (Cell Signaling, 8828), hnRNP-K (Cell Signaling, 4675S), E-cadherin (Cell Signaling, 5296S), and MMP2 (Santa Cruz, SC-13594) proteins were used for immunostaining or blotting as indicated.

Techniques: Western Blot, Immunostaining

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) Domain architecture of HP1α and HP1γ: nte, amino-terminal extension; CD, chromodomain; CSD, chromoshadow domain; cte, carboxyl-terminal extension. (B) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled HP1γ CSD recorded in the presence of increasing amounts of KAP1 Hbox1 peptide. Spectra are color coded according to the protein:peptide molar ratio. (C) Binding affinities of wild type or mutated HP1γ CSD for the indicated KAP1 peptides. (a) from Gaurav et al. and (b) determined by NMR. (D) Representative binding curve used to determine the binding affinity of HP1γ CSD for KAP1 Hbox by tryptophan fluorescence. K d is represented as the average ± SD of three independent experiments. n = 3. (E and F) The crystal structure of the HP1γ CSD dimer in complex with KAP1 Hbox peptide. Two protomers of the HP1γ CSD dimer are shown in a ribbon diagram and labeled CSD1 and CSD2. KAP1 Hbox is depicted as a ribbon in (E) or sticks in (F). (G) A close view of the KAP1 Hbox -binding site of the HP1γ CSD dimer. Residues involved in the interaction between HP1γ CSD and KAP1 Hbox are labeled. CSD2 residues are labeled with the apostrophe. Dashed lines represent hydrogen bonds. (H and J) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled wild-type or mutated HP1γ CSD recorded in the presence of increasing amounts of wild-type or mutated KAP1 Hbox peptide. Spectra are color coded according to the protein:peptide molar ratio. See also .

Article Snippet: HP1γ hinge (aa 81–108) , SynPeptide , N/A.

Techniques: Labeling, Binding Assay, Fluorescence

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled HP1γ CSD (purple) and FL HP1γ. The FL HP1γ spectra were recorded in the presence of increasing amounts of KAP1 Hbox peptide and are color coded according to the protein:peptide molar ratio. HP1γ CSD resonances in the spectrum of FL HP1γ broadened beyond detection, indicating that the size of this part of HP1γ becomes too large, i.e., HP1γ CSD multimerization (higher order than detectable dimerization) reduces the tumbling rate. (B) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled S95D/S97D and Δhinge mutants of FL HP1γ recorded in the presence of increasing amounts of KAP1 Hbox peptide. (C) Superimposition of the crystal structure of one protomer of HP1γ CSD (wheat) in complex with KAP1 Hbox (green) and the AlphaFold model of FL HP1γ (gray, with CD and cte colored pink and light cyan, respectively, from UniProt, # Q13185 ). (D) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled I165E HP1γ CSD recorded in the presence of increasing amounts of unlabeled wild-type HP1γ CSD . Spectra are color coded according to the protein:ligand molar ratio. (E) Molecular mass distribution histograms of FL HP1γ at indicated concentrations in mass photometry assay. Maxima of the fits are labeled (kDa). (F) Molecular mass distribution histogram of the 2:1 mixture of FL HP1γ and KAP1 Hbox1 peptide in mass photometry assay. Maxima of the fits are labeled (kDa). (G) Two KAP1 Hbox -bound dimers of HP1γ CSD (one dimer is colored wheat, and the other is colored light blue) interact via their β-sheet regions. KAP1 Hbox is shown as green ribbon and, due to symmetry, can be bound in either direction. The α-helix interface and β-sheet interface in the tetrameric organization of the complex are labeled.

Article Snippet: HP1γ hinge (aa 81–108) , SynPeptide , N/A.

Techniques: Labeling

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) Superimposition of the crystal structure of the KAP1 Hbox -bound dimer of HP1γ CSD (wheat) with two molecules of the AlphaFold model of FL HP1γ (one FL HP1γ is colored gray, and the other FL HP1γ is colored magenta). Regions of FL HP1γ are labeled. (B) Two peripheral protomers from the KAP1 Hbox -bound dimer of dimers of HP1γ CSD (colored as in ) are superimposed with two molecules of the AlphaFold model of FL HP1γ (one FL HP1γ is colored gray, and the other FL HP1γ is colored magenta). (C–E) Overlaid 1 H, 15 N HSQC spectra of 15 N-labeled HP1γ CSD recorded in the presence of increasing amounts of HP1γ CD (C), HP1γ hinge (D), or HP1γ cte (E). Spectra are color coded according to the protein:peptide molar ratio.

Article Snippet: HP1γ hinge (aa 81–108) , SynPeptide , N/A.

Techniques: Labeling

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) A close view of the β-sheet interface between two KAP1 Hbox -bound dimers of HP1γ CSD (colored as in ). Only one protomer per dimer is shown for clarity. KAP1 Hbox is depicted as green ribbon. The residues involved in the formation of the β-sheet interface are shown as sticks and labeled. (B) Molecular mass distribution histograms of K141E HP1γ (top), R125E/K141E HP1γ (middle), and the 1:3 mixture of R125E/K141E HP1γ and KAP1 Hbox (bottom) in mass photometry assay. Maxima of the fits are labeled (kDa). (C) Molecular mass distribution histograms of HP1α at indicated concentrations in mass photometry assay. Maxima of the fits are labeled (kDa). (D) A close view of the β-sheet interface between two KAP1 Hbox ′-bound dimers of HP1α CSD (Gaurav et al. ). KAP1 Hbox ′: aa 468–496 of KAP1. Only one protomer per dimer is shown for clarity. KAP1 Hbox is depicted as a yellow ribbon. The residues involved in the formation of the β-sheet interface are shown as sticks and labeled. (E) The crystal structures of dimers of the KAP1 Hbox -bound dimers of HP1γ and HP1α CSD are superimposed. Two protomers of one HP1γ CSD dimer (CSD1 and CSD2) are colored wheat, and two protomers of the second HP1γ CSD dimer (CSD3 and CSD4) are colored light blue. Both dimers of HP1α CSD are colored green. The dimers interact through their β-sheets. (F) A model of limited multimerization of HP1γ CSD and robust unlimited multimerization of HP1α CSD . (G) Bright-field images of concentration series of WT HP1γ, WT HP1α, and L139E/L150E HP1α with 30 nM of 2.7 kbp linearized PUC19 plasmid DNA. Scale bar represents 20 μm.

Article Snippet: HP1γ hinge (aa 81–108) , SynPeptide , N/A.

Techniques: Labeling, Concentration Assay, Plasmid Preparation

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) AI662270 expression was measured in HP1α −/− , HP1β −/− , HP1γ −/− , and KAP1 −/− mouse ESCs using quantitative reverse transcription PCR (qRT-PCR). Data are presented as average between two independent biological replicates. (B) Generation of the entry mouse ESC line. The attP sequence (MIN tag) is inserted directly after the transcription start site of HP1γ. Schematic illustrating the CRISPR-Cas9-mediated genome editing strategy, with the gRNA and PAM sequences highlighted. The donor single-strand DNA contains the MIN tag sequence with a HincII restriction cut site for screening and homology arms flanking the translational start site. The positions of screening PCR primers are indicated, which produce 509 and 557 bp products in WT and HP1γ attP/attP cells, respectively. (C) PCR-based validation of two individual HP1γ attP/attP ESCs using primers indicated in (B), followed by HincII digestion. PCR products showing a reduced size after HincII digestion are considered positive. (D) Schematic outlining the Bxb1-mediated recombination strategy used to generate the HP1γ −/− ESC line. A cassette containing the attB site, RFP, a stop codon, and a polyA signal was inserted directly after the transcription start site by attP-attB recombination to produce HP1γ −/− ESCs. (E and F) Characterization of two individual HP1γ −/− ESC clones, A3 and C3, by quantitative RT-PCR analysis (E). Data are presented as mean ± standard deviation (SD) from three technical replicates. Western blot analysis to detect HP1γ in the two individual HP1γ −/− clones (F). The tubulin blot was used as a loading control. See also 1 and .

Article Snippet: HP1γ hinge (aa 81–108) , SynPeptide , N/A.

Techniques: Expressing, Reverse Transcription, Quantitative RT-PCR, Sequencing, CRISPR, Biomarker Discovery, Clone Assay, Standard Deviation, Western Blot, Control

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) Expression levels of AI662270 were measured by quantitative reverse transcription PCR (RT-PCR) in KAP1 −/− ESCs stably expressing GFP-tagged KAP1 fragment (aa 114–834 of KAP1, GFP-KAP1 FR ) (WT or the V488E mutant). The expression data in rescue cell lines were normalized to the protein levels of GFP-KAP1 FR and are presented as mean ± SEM from three independent biological replicates. n = 3. Statistical analyses were performed using an unpaired, two-tailed Student’s t test; *, **, and **** stand for p values < 0.05, 0.01, and 0.0001, respectively. (B) Representative immunofluorescence images show the cellular distribution of GFP-KAP1 FR WT and the V488E mutant stably expressed in KAP1 −/− ESCs. Endogenous HP1γ was visualized by immunostaining with an anti-HP1γ antibody. (C and D) Boxplots show the variation of coefficient for GFP-KAP1 FR intensity (C) and the Pearson correlation coefficient between GFP-KAP1 FR and endogenous HP1γ (D). Statistical analyses were performed using an unpaired two-tailed Student’s t test; **** stands for p value < 0.0001. (E) Expression levels of AI662270 were measured by quantitative RT-PCR in HP1γ −/− ESCs stably expressing GFP-tagged HP1γ (WT or the W174A mutant). The expression data in rescue cell lines were normalized to the protein levels of GFP-HP1γ and are presented as mean ± SEM from three independent biological replicates. n = 3. Statistical analyses were performed using an unpaired two-tailed Student’s t test; *, **, and **** stand for p values < 0.05, 0.01, and 0.0001, respectively. (F) Representative immunofluorescence images show the cellular distribution of GFP-HP1γ WT and the W174A mutant (together with mCherry-KAP1 FR ) stably expressed in HP1γ −/− ESCs. (G and H) Boxplots show the variation of coefficient for GFP-HP1γ intensity (G) and the Pearson correlation coefficient between GFP-HP1γ and mCherry-KAP1 FR (H). Statistical analyses were performed using an unpaired, two-tailed Student’s t test; *** and **** stand for p values < 0.001 and 0.0001, respectively. (I) Genome browser representation of enrichments of HP1γ and KAP1 ChIP-seq in WT ESCs and H3K9me3 CUT&Tag in WT and KAP1 KO ESCs at AI662270 . The y axis represents read density in counts per million mapped reads (CPM). Putative AI662270 enhancers were identified using the ABC model. (J) The expression of AI662270 in NPCs derived from WT, KAP1 −/− and HP1 −/− NPCs by quantitative RT-PCR analyses. Data are presented as mean ± SD, based on two biological replicates for HP1 knockout cells and four technical replicates for WT and KAP1 −/− cells. Statistical analyses were performed using an unpaired, two-tailed Student’s t test. *, **, and *** indicate p values < 0.05, 0.01, and 0.001, respectively, while n.s. denotes no significant difference. See also .

Article Snippet: HP1γ hinge (aa 81–108) , SynPeptide , N/A.

Techniques: Expressing, Reverse Transcription, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Mutagenesis, Two Tailed Test, Immunofluorescence, Immunostaining, Quantitative RT-PCR, ChIP-sequencing, Derivative Assay, Knock-Out

Journal: Cell reports

Article Title: HP1γ self-assembles and cooperates with KAP1 in repression of long noncoding RNA AI662270 in ESCs

doi: 10.1016/j.celrep.2025.116874

Figure Lengend Snippet: (A) AI662270 expression was measured in mouse tissues using quantitative RT-PCR. Data are presented as mean ± SD from four technical replicates. n = 4. Statistical analyses were performed using an unpaired, two-tailed Student’s t test to compare HP1γ with HP1α and HP1β. *, **, and *** indicate p < 0.05, 0.01, and 0.001, respectively, while n.s. denotes no significant difference. (B) A heatmap illustrating the expression profiles of shared dysregulated genes in HP1 knockout cells, with expression levels indicated by color intensity. (C) Gene Ontology (GO) analysis of shared dysregulated genes in HP1α −/− and HP1γ −/− mouse ESCs. (D and E) Gene Ontology (GO) analysis of up- and downregulated genes in KAP1 −/− mouse ESCs.

Article Snippet: HP1γ hinge (aa 81–108) , SynPeptide , N/A.

Techniques: Expressing, Quantitative RT-PCR, Two Tailed Test, Knock-Out