Journal: PLoS ONE

Article Title: Bortezomib Reduces the Tumorigenicity of Multiple Myeloma via Downregulation of Upregulated Targets in Clonogenic Side Population Cells

doi: 10.1371/journal.pone.0056954

Figure Lengend Snippet: (A). Real time quantitative PCR of cell cycle and mitosis related genes ( CCNB1,CDC2, CDC20, CDC25C, AURKB, BIRC5, TOP2A, ASPM), Polycomb related genes ( EPC1, EZH2), and ubiquitin-proteasome related gene ( UBE2D3 and PSMA5) against RPMI 8226, AMO1, KMS-12-BM, JJN3 and KMS-11 cells. Y-axis: gray and white bars depict 2 −ΔΔCt values for gene expression. Asterisks (*) indicate statistical significance: *0.01≤ P <0.05, **0.001≤ P <0.01, *** P <0.001. Bars are means ± SD of three independent experiments. (B). Western blot analysis of Cyclin B1, CDC2, p-WEE1, p-CDC2, Aurora B, p-Aurora B, p-Hist.H3, EZH2, PSMA5 and GAPDH in SP and MP against RPMI 8226 and AMO1 cell lines.

Article Snippet: TaqMan probes of CCNB1 (Hs01030097_m1), EZH2 (Hs01016789_m1), TOP2A (Hs00172214_m1), CDC2 (Hs00938777_m1), CDC20 (Hs00415851_g1), CDC25C (Hs00156411_m1), ASPM (Hs00411505_m1), AURKB (Hs00177782_m1), BIRC5 (Hs00220565_m1), UBE2D3 (Hs00704312_m1), PSMA5 (Hs00936004_m1), EPC1 (Hs00228677_m1) and GAPDH (Hs02758991_g1) were purchased from Applied Biosystems.

Techniques: Real-time Polymerase Chain Reaction, Ubiquitin Proteomics, Gene Expression, Western Blot

Journal: PLoS ONE

Article Title: Bortezomib Reduces the Tumorigenicity of Multiple Myeloma via Downregulation of Upregulated Targets in Clonogenic Side Population Cells

doi: 10.1371/journal.pone.0056954

Figure Lengend Snippet: (A). SP of primary samples (M4, M7 and M8). Left panel: cells obtained through bone marrow aspiration gated for CD138 + with and without 50 µM reserpine. SP fractions (%) are shown beside the SP gates surrounded by black lines. (B). Real time PCR analysis of eight samples of MM primary tumor cells. Shown bar graphs are CCNB1, EZH2, AURKB and PSMA5 in SP and MP cells of indicated five myeloma cell lines. Y-axis: gray and white bars depict 2 −ΔΔCt values for gene expression. Asterisks (*) indicate statistical significance: *0.01≤ P <0.05, **0.001≤ P <0.01, *** P <0.001. Bars are means ± SD of triplicate samples.

Article Snippet: TaqMan probes of CCNB1 (Hs01030097_m1), EZH2 (Hs01016789_m1), TOP2A (Hs00172214_m1), CDC2 (Hs00938777_m1), CDC20 (Hs00415851_g1), CDC25C (Hs00156411_m1), ASPM (Hs00411505_m1), AURKB (Hs00177782_m1), BIRC5 (Hs00220565_m1), UBE2D3 (Hs00704312_m1), PSMA5 (Hs00936004_m1), EPC1 (Hs00228677_m1) and GAPDH (Hs02758991_g1) were purchased from Applied Biosystems.

Techniques: Real-time Polymerase Chain Reaction, Gene Expression

Journal: PLoS ONE

Article Title: Bortezomib Reduces the Tumorigenicity of Multiple Myeloma via Downregulation of Upregulated Targets in Clonogenic Side Population Cells

doi: 10.1371/journal.pone.0056954

Figure Lengend Snippet: (A). Cell cycle analysis of RPMI 8226 and AMO1 cells exposed toVX-680 (1 µM). X-axis, PI; Y-axis, cell count. RPMI 8226+DMSO: subG1 1.1%, G 0 /G 1 47.3%, S 18.5%, G 2 /M 33.2%; RPMI 8226+VX-680 (1 uM): subG 1 1.7%, G 0 /G 1 3.4%, S 16.4%, G 2 /M 78.5%. AMO1+DMSO: subG 1 1.9%, G 0 /G 1 82.5%, S 22.4%, G 2 /M 12.9%; AMO1+VX-680 (1 uM): subG 1 8.4%, G 0 /G 1 54.8%, S 10.5%, G 2 /M 30.7%. (B). Detection of M phase cells among VX-680-treated MM cells. Upper panels: DAPI and p-Hist.H3 staining (green) of cells treated with DMSO, 1 µM, and 10 µM VX-680 (24 hr exposure). Under panels: bar graphs showing the numbers of M phase cells after treatment with the indicated concentration of VX-680 (24 hr exposure). (C). Western blot analysis of p-Hist.H3, EZH2 in RPMI 8226 (left panel) and AMO1 (right panel) cells; Tubulin is the control. (D). Flow cytometric analysis of RPMI 8226 SP cells. Dot plots of cells stained with Hoechst 33342 alone, Hoechst 33342 in the presence of 1 µM VX-680 or Hoechst 33342 in the presence of 10 µM VX-680. Left upper panels: 24 h exposure to VX-680; left lower panels: 48 h exposure to VX-680. Bar graphs of SP cell fractions (%) of indicated cells treated with VX-680 (DMSO, 1 µM, 10 µM) for 24 hr or 48 hr are also shown besides the flow cytometric analysis. DMSO is the control. Asterisks (*) indicate statistical significance: *0.01≤ P <0.05, **0.001≤ P <0.01, *** P <0.001. Bars are means ± SD of triplicate samples. (E). CFC assay. Colonies of SP by VX-680 (DMSO, 1 µM, 10 µM) for RPMI 8226 and AMO1 cell lines. Colony count was examined after 10 days from SP or MP distribution.

Article Snippet: TaqMan probes of CCNB1 (Hs01030097_m1), EZH2 (Hs01016789_m1), TOP2A (Hs00172214_m1), CDC2 (Hs00938777_m1), CDC20 (Hs00415851_g1), CDC25C (Hs00156411_m1), ASPM (Hs00411505_m1), AURKB (Hs00177782_m1), BIRC5 (Hs00220565_m1), UBE2D3 (Hs00704312_m1), PSMA5 (Hs00936004_m1), EPC1 (Hs00228677_m1) and GAPDH (Hs02758991_g1) were purchased from Applied Biosystems.

Techniques: Cell Cycle Assay, Cell Counting, Staining, Concentration Assay, Western Blot, Control

Journal: PLoS ONE

Article Title: Bortezomib Reduces the Tumorigenicity of Multiple Myeloma via Downregulation of Upregulated Targets in Clonogenic Side Population Cells

doi: 10.1371/journal.pone.0056954

Figure Lengend Snippet: (A). Frequency of apoptosis of RPMI 8226 and AMO1. Left panels: dot plots showing the frequency of apoptosis at the indicated bortezomib (Bor.) concentrations (48 hr exposure). X-axis: cells stained with AnnexinV-PE. Y-axis: cells stained with 7-AAD. Right panels: bar graphs showing the % apoptotic cells (R1+R2) among examined cells treated with indicated concentration of bortezomib at 24 hr and 48 hr as indicated. Asterisks (*) indicate statistical significance: *0.01≤ P <0.05, **0.001≤ P <0.01, *** P <0.001. NS: not significant. (B). Cell cycle analysis RPMI 8226 and AMO1 treated with 10 nM bortezomib (24 hr). DMSO served as the control. RPMI 8226 control (+DMSO): subG 1 3.9%, G 0 /G 1 48.3%, S 19.2%, G 2 /M 28.6%; RPMI 8226+ bortezomib (10 nM): subG 1 5.0%, G 0 /G 1 20.1%, S 20.4%, G 2 /M 54.4%. AMO1 control (+DMSO): subG 1 1.3%, G 0 /G 1 58.4%, S 18.5%, G 2 /M 21.7%; AMO1+ bortezomib (10 nM): subG 1 14.8%, G 0 /G 1 31.6%, S 23.6%, G 2 /M 29.6% (C). Detection of M phase cells among bortezomib-treated (48 hr) myeloma cells. Bar graphs showing the numbers of M phase cells after treatment with DMSO, 1 nM, 10 nM and 100 nM bortezomib (24 hr exposure.) (D).Western blot analysis of p-Hist.H3 and EZH2 in RPMI 8226 (left panel) and AMO1 (right panel) cells after treatment with the indicated concentration of bortezomib and dexamethasone (48 hr). (E). Flow cytometric analysis of RPMI 8226 SP cells treated with bortezomib. Upper left panels: Dot plots of cells stained with Hoechst 33342 alone, Hoechst 33342 in the presence of 1 nM bortezomib for 48 h, or Hoechst 33342 in the presence of 10 nM bortezomib for 48 h. Lower left panels: cells treated as in the upper panels with 50 µM reserpine (shown as “res”). SP cell fractions (%) after treating RPMI 8226 and AMO1 cells are also shown besides the flow cytometric analysis. (F). CFC assay. Colonies of SP and MP by dexamethasone (left panel, Control (1 µl of 100% ethanol), 0.1 µM, 1 µM) and bortezomib (right pane, DMSO, 1 nM or 10 nM) for indicated cell lines. Asterisks (*) indicate statistical significance: *0.01≤ P <0.05, **0.001≤ P <0.01, *** P <0.001. Bars are means ± SD of three independent experiments.

Article Snippet: TaqMan probes of CCNB1 (Hs01030097_m1), EZH2 (Hs01016789_m1), TOP2A (Hs00172214_m1), CDC2 (Hs00938777_m1), CDC20 (Hs00415851_g1), CDC25C (Hs00156411_m1), ASPM (Hs00411505_m1), AURKB (Hs00177782_m1), BIRC5 (Hs00220565_m1), UBE2D3 (Hs00704312_m1), PSMA5 (Hs00936004_m1), EPC1 (Hs00228677_m1) and GAPDH (Hs02758991_g1) were purchased from Applied Biosystems.

Techniques: Staining, Concentration Assay, Cell Cycle Assay, Control, Western Blot

Journal: Frontiers in Oncology

Article Title: Histone Modifications Drive Aberrant Notch3 Expression/Activity and Growth in T-ALL

doi: 10.3389/fonc.2019.00198

Figure Lengend Snippet: GSKJ4 and A-485 treatments modulate Notch receptors expression and activity. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (A) TALL-1 or (C) MOLT3 cells treated for 48 h with 2 μM GSKJ4 or with DMSO. (B) Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panel) and HA and β-actin protein levels (lower panel) in TALL-1 cells transfected with HA-tagged EZH2 expression vector (HA-EZH2) or with the empty control vector. Relative NOTCH1, NOTCH3 , and DELTEX1 gene expression (upper panels) and N1ICD, N3ICD, β-actin, H3K27me3, H3K27ac, and H3 total expression levels (lower panels) in: (D) TALL-1 or (E) MOLT3 cells treated for 48 h with 5 μM A-485 or DMSO. Data represent mean values of three biological replicates ± Standard Error of the Mean (S.E.M.); ( n = 3) * P < 0.05, ** P < 0.01, *** P < 0.001. Uncropped western blots related to this figure are displayed in .

Article Snippet: The expression vector PIRVNeoSV containing the human c-Myc cDNA coding sequence (c-Myc) was kindly provided by Dr. Giuseppe Giannini (Sapienza University, Rome, Italy). pCMV3-HA vector containing the human EZH2 coding sequence (HA-EZH2) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Western Blot

Journal: PLoS Biology

Article Title: Polycomb Repressive Complex 2 (PRC2) Restricts Hematopoietic Stem Cell Activity

doi: 10.1371/journal.pbio.0060093

Figure Lengend Snippet: (A) A schematic representation of part of the Suz12 locus is shown to detail the intron and exon structure (at left). Primers were designed to flank the site of the mutation (red arrow), and RT-PCR was performed on cDNA prepared from bone marrow. An aberrantly spliced product (asterisk) was identified in cDNA prepared from Suz12 Plt8/+ mice that was not present in wild type. (B) Protein lysates were prepared from sex-matched embryonic day 12.5 (E12.5) embryos for analysis by Western blotting, which revealed that Suz12 and Ezh2 protein levels were reduced in Suz12 Plt8/+ embryos. Suz12 protein levels appeared equivalent in Suz12 Plt8/+ embryos and embryos heterozygous for the genetrap allele ( Suz12 502gt/+ ). Equivalent amounts of protein were run in each lane (20 μg), and histone H3 was used to verify equal loading. Western blot signal intensity was quantified using a densitometer; results represent the average of two independent experiments expressed as protein expression in Suz12 Plt8/+ (gray) and Suz12 502gt/+ (black) embryos relative to wild type (white, 100%).

Article Snippet: Protein was transferred to a PVDF membrane and blotted with antibodies to detect Suz12, Ezh2, Histone 3, H3K27-3Me (Upstate), Akt (Cell Signaling) or the FLAG-epitope (M2) (Sigma).

Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing

Journal: PLoS Biology

Article Title: Polycomb Repressive Complex 2 (PRC2) Restricts Hematopoietic Stem Cell Activity

doi: 10.1371/journal.pbio.0060093

Figure Lengend Snippet: Bone marrow extracted from 5-FU–treated mice was infected with either the LMS-Nons or the LMS-Suz12 virus and transplanted into recipient mice. Three independent infections were performed, and in each case, infected cells were transplanted into five recipient animals. A selection of primary recipients (9–11) were used as donors for secondary transplants, in each case these cells were transplanted into 3–5 recipient mice. (A) Thymocytes were isolated from primary recipients 12 wk after transplantation and fractionated based upon expression of GFP (+ or –); low or intermediate populations were detected in some mice (low). Protein lysates were prepared from sorted cells and Western blotting was performed to detect expression of Suz12, Ezh2, or histone H3. Nonspecific bands have been marked (*) and an arrow is used to denote residual Suz12 signal that persisted after the membrane was stripped and reprobed. (B) The frequency of cells that carried the virus (GFP + ) was monitored prior to transplantation (Input) and at 8–12 wk after transplantation in primary or secondary recipients. (C) The representation of GFP + cells was compared between donor and recipient populations and a ratio calculated (recipient GFP%/donor GFP%). Equal representation in recipient and donor populations would result in a ratio of 1.0. The representation of cells infected with LMS-Suz12 continued to increase over the course of the experiment, whereas the representation of LMS-Nons cells remained constant. Data show the mean and standard error. Statistical significance was assessed using an unpaired t -test.

Article Snippet: Protein was transferred to a PVDF membrane and blotted with antibodies to detect Suz12, Ezh2, Histone 3, H3K27-3Me (Upstate), Akt (Cell Signaling) or the FLAG-epitope (M2) (Sigma).

Techniques: Infection, Virus, Selection, Isolation, Transplantation Assay, Expressing, Western Blot, Membrane

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: CDYL enhances PRC2 activity in vitro. A, Coomassie Blue staining of PRC2 complexes (containing EZH2, SUZ12, and EED) purified from Sf9 cells. B, MNase digestion of reconstituted oligonucleosomes resolved by 2% agarose gel. Left panel: lane 1 shows the pure pG5E4 plasmid DNA, and lane 2 shows band shift of pG5E4 DNA assembly into oligonucleosomes. Right panel: equimolar amounts of reconstituted oligonucleosomes were digested with increasing amounts of MNase (Sigma). The DNA was isolated and subjected to electrophoresis on a 2% agarose gel in the presence of ethidium bromide. Partial MNase digestion (oligonucleosome: MNase = 2 μg: 0.5 μl) generated a nucleosomal DNA ladder with visible mono-, di-, and trinucleosomal fragments, which are indicated by corresponding numbers of asterisks. Mononucleosomal DNA runs as a 147 bp fragment. C, CDYL stimulates PRC2 activity in vitro. Reconstituted recombinant oligonucleosomes were incubated with EZH2/SUZ12/EED complexes (PRC2-core) in the absence or presence of increasing amounts of baculovirus generated CDYL and histone methyltransferase activity was determined by standard HMT assays. The reaction products were examined by Western blotting with the antibodies indicated on the right. Ponceau staining of histones is shown in the bottom panel to show equal amounts of substrates used in each reaction. D, CDYL only stimulates PRC2 methyltransferase activity toward oligonucleosome, but not mononucleosome substrates. Reconstituted Xenopus oligonucleosomes were digested with MNase (oligonucleosome: MNase = 1 μg: 1 μl) at room temperature for 5 min. This treatment yielded mainly mononucleosomes (see Fig. 4B). Equal amounts of mononucleosomes were used as substrates for the HMT assay in the top panel, whereas equal amounts of undigested oligonucleosomes were used as substrates in the bottom panel. Commercially available EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (PRC2-full) were used to provide methyltransferase activity as indicated. The mild increase of PRC2 activity seen upon CDYL addition in the top panel was mainly due to incomplete digestion of oligonucleosomes (see Fig. 4B). E, binding affinity between CDYL and H3K27me3 is much stronger than the affinity between EED and H3K27me3. In the top panel, histone peptide binding assays show that CDYL, but not EED, binds to H3K27me2 when the same amounts of FLAG-tagged proteins (0.5 μg) were used in the assay. To compare the binding affinity for H3K27me3, 0.2, 0.4, or 1 μg of baculovirus-expressed FLAG-CDYL and 0.6, 1.2, or 3 μg of FLAG-EED proteins were used in the peptide binding assay. Ten percent of total proteins were used as loading controls (middle panel). Peptide-protein complexes were pulled down by streptavidin beads and bound proteins were examined by Western blotting using anti-FLAG antibodies (bottom panel). Ponceau staining of baculovirus-expressed FLAG-CDYL and FLAG-EED is shown in the right panel.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: Activity Assay, In Vitro, Staining, Purification, Agarose Gel Electrophoresis, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Isolation, Electrophoresis, Generated, Recombinant, Incubation, Western Blot, HMT Assay, Binding Assay

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: CDYL is physically associated with the PRC2 complex. A, in vivo immunoprecipitation. Top two panels: endogenous IP of MCF-7 cell lysates using antibodies against CDYL. Antibodies (EZH2 or SUZ12) used for Western blotting are indicated on the right. Bottom panel: MCF-7 cells were transfected with a FLAG-CDYL construct and subjected to co-IP assays after 48 h. Cell protein extracts were immunoprecipitated with polyclonal antibodies against EED, and blotted with monoclonal anti-FLAG antibodies. An immunoglobulin G (IgG) control is included in each experiment. B, GST pull-down assays. Purified GST or GST-CDYL proteins immobilized on glutathione Sepharose 4B beads were incubated with in vitro translated EZH2, SUZ12, or EED. Bound proteins were detected with monoclonal anti-EZH2 antibodies (top panel) or anti-MYC tag antibodies (lower two panels). C, Superose 6 gel filtration analysis of the MCF-7 nuclear extracts. Migration of molecular markers is indicated above the panels and the antibodies for Western blotting are indicated on the right. Equal volumes from each fraction were analyzed. D, similar FPLC experiments as in C using a Superdex 200 10/300 GL column. The chromatographic fractions were analyzed by Western blotting using the indicated antibodies. Bottom panel: HMT assays were performed using the indicated eluted fractions. Recombinant Xenopus H3 proteins were used as substrates, and the reaction products were analyzed by Western blotting with anti-H3K27me3 antibodies.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: In Vivo, Immunoprecipitation, Western Blot, Transfection, Construct, Co-Immunoprecipitation Assay, Purification, Incubation, In Vitro, Filtration, Migration, Recombinant

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: Mapping the domains responsible for the interaction between CDYL and EZH2. A, schematic drawing of CDYL protein. CDYL deletion mutants including del1 (1–309 aa), del2 (1–60 aa, the chromodomain), del3 (61–545 aa), del4 (310–545 aa, the coAP domain), and del5 (61–309 aa) were fused to GST. B, GST pull-down experiments were performed with in vitro translated FLAG-EZH2 and purified GST or GST-CDYL deletion mutants. The precipitated complexes were examined by Western blotting using monoclonal anti-EZH2 antibodies (the upper panel). Only CDYL mutants containing the middle region from 61–309 aa (del1, del3, del5) efficiently pulled down EZH2. The lower panel shows the Ponceau staining of purified GST fusion proteins added to the reaction. The arrows indicate the positions of the respective GST fusion proteins as labeled on the top. C, schematic drawing of EZH2 protein. EZH2 deletion mutants (del1 to del5) were cloned into the pGBKT7 plasmid, which contains a c-Myc epitope tag and can be transcribed/translated in vitro. Del8 and Del9 were fused to GST. D, GST pull-down experiments were performed with in vitro translated Myc-EZH2 deletion mutants and purified GST or GST-CDYL in the left panels. Right panel: GST pull-down assays were performed with in vitro translated Myc-CDYL, which was incubated with purified GST, GST-del8, GST-del9, or GST-SET8 (negative control protein). Bound proteins were examined by Western blotting using monoclonal anti-Myc antibodies.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: In Vitro, Purification, Western Blot, Staining, Labeling, Clone Assay, Plasmid Preparation, Incubation, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27 *

doi: 10.1074/jbc.M111.271064

Figure Lengend Snippet: Validation of common target genes of CDYL and PRC2. A, quantitative ChIP assays were performed in MCF-7 cells with primer pairs specific to indicated gene promoters (see supplemental Table S1). Normal rabbit IgG, as well as polyclonal antibodies against CDYL, EZH2, and H3K27me3 were used to immunoprecipitate the protein-DNA complex. B, conventional semi-quantitative ChIP assays performed at the MYT1 and BASE promoters. C, CDYL and PRC2 exist in the same protein complex at the MYT1 and BASE promoters. ChIP and re-ChIP experiments were performed with the indicated antibodies and primer pairs. D, CDYL expression was efficiently knocked down by specific siRNAs. Non-silencing or CDYL specific siRNAs were transfected into MCF-7 cells. Total proteins were extracted and the expression of CDYL and EZH2 proteins were examined by Western blotting. Actin protein levels were measured to indicate equal loading of protein lysates. E, CDYL is required for PRC2 chromatin targeting at the MYT1 and BASE promoters. MCF-7 cells were transfected with control siRNA or CDYL-specific siRNA. 48 hours after the transfection, cell lysates were collected, and ChIP experiments were performed using the indicated antibodies. Real-time PCR assays were performed for the measurement. F, CDYL mainly represses the expression of target genes. MCF-7 cells were transfected with control or CDYL-specific siRNAs. Total RNAs were prepared and the mRNA levels of the indicated genes were examined by real-time RT-PCR. The data were normalized against the expression of GAPDH. Each bar represents the mean ± S.D. for triplicate measurements.

Article Snippet: For D , 0.5 μg of recombinant human EZH2/EED/SUZ12/RbAp48/AEBP2 complexes (BPS Bioscience catalogue no. 51004) were used to provide methyltransferase activity and 2 μg of recombinant oligonucleosomes or mononucleosomes were used as substrates.

Techniques: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Oncology Letters

Article Title: miR-26a inhibits invasion and metastasis of nasopharyngeal cancer by targeting EZH2

doi: 10.3892/ol.2013.1173

Figure Lengend Snippet: EZH2 was inversely correlated with miR-26a levels. (A) The expression levels of miR-26a and EZH2 in 5-8F cells transfected with LV-control and LV-miR-26a. ** P<0.01 compared with the control group. (B) The expression of EZH2 protein in cells transfected with LV-miR-26a was decreased compared with the control. (C) Immunohistochemistal staining of EZH2 in primary liver tumor tissues of NPC metastasis-bearing mice. The representative images are presented (magnification, ×100). EZH2, enhancer of zeste homolog 2; NPC, nasopharyngeal carcinoma.

Article Snippet: The membrane was incubated with a rabbit monoclonal antibody against human EZH2 (1:500 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA) followed by HRP-labeled goat anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and detected by chemiluminescence.

Techniques: Expressing, Transfection, Control, Staining

Journal: Oncology Letters

Article Title: miR-26a inhibits invasion and metastasis of nasopharyngeal cancer by targeting EZH2

doi: 10.3892/ol.2013.1173

Figure Lengend Snippet: Immunohistochemical detection of EZH2 in primary tumors in the control and miR-26a groups.

Article Snippet: The membrane was incubated with a rabbit monoclonal antibody against human EZH2 (1:500 dilution, Cell Signaling Technology, Inc., Danvers, MA, USA) followed by HRP-labeled goat anti-mouse IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and detected by chemiluminescence.

Techniques: Immunohistochemical staining, Control

Journal: Genes, Brain, and Behavior

Article Title: Maternal upbringing and selective breeding for voluntary exercise behavior modify patterns of DNA methylation and expression of genes in the mouse brain

doi: 10.1111/gbb.12858

Figure Lengend Snippet: Coordinates, genomic context and number of CpG sites analyzed for 14 genes analyzed by bisulfite sequencing.

Article Snippet: Ezh2 : enhancer of zeste homolog 2 , Mm00468464_m1 , Chr6: 47530274–47595340 , 19 and 20.

Techniques: Sequencing

Journal: Genes, Brain, and Behavior

Article Title: Maternal upbringing and selective breeding for voluntary exercise behavior modify patterns of DNA methylation and expression of genes in the mouse brain

doi: 10.1111/gbb.12858

Figure Lengend Snippet: List of genes and the corresponding TaqMan assays used for RT‐qPCR.

Article Snippet: Ezh2 : enhancer of zeste homolog 2 , Mm00468464_m1 , Chr6: 47530274–47595340 , 19 and 20.

Techniques: TaqMan Assay, Binding Assay

Journal: Theranostics

Article Title: HOXB13 networking with ABCG1/EZH2/Slug mediates metastasis and confers resistance to cisplatin in lung adenocarcinoma patients

doi: 10.7150/thno.29463

Figure Lengend Snippet: HOXB13 targets to and upregulates EZH2. (A) Upregulation of EZH2 by HOXB13 in lung adenocarcinoma cells. H1299 and A549 cells were transiently transfected by Flag-HOXB13 or GFP-HOBX13 separately, controlled by Flag or GFP. Left panel: Cell lysates were prepared and were subjected to Western blot analysis using anti-EZH2 antibody. Right panel: Transcriptional detection of HOXB13-upregulated EZH2 by qPCR. (B) Enrichment of HOXB13 on the EZH2 promoter analyzed by ChIP-seq database from prostate cancer . (C) HOXB13 targets EZH2 in lung adenocarcinoma cells. Upper panel: Diagram of the EZH2 promoter with potential HOXB13 binding sites (double arrow). Lower panel: ChIP analysis was performed using either an anti-HOXB13 ChIP-grade antibody or control IgG in H1299 Flag-HOXB13 cells. Sites 3, 4, and 5 in EZH2 promoter are enriched in a qPCR analysis with known target genes of HOXB13 including ORM1, NKX3.1 as positive controls, and actin as a negative control. Insert is the gel picture of ChIP analysis for HOXB13 targeting on EZH2 promoter. (D) EZH2 promoter-luciferase reporter construct map. Lower panel: Luciferase reporter constructs were co-transfected with vector or HOXB13, towards the identification of 1062-1875bp upstream region critical for HOXB13-directed enhancement (Unpaired Student's t -test, **p < 0.01) in H1299 (left panel) and in A549 cells (right panel). (E) Levels of HOXB13 and EZH2 in patients' tumor specimens were detected by immunohistochemical analyses using HOXB13 and EZH2 antibodies separately. Patients 1-3: HOXB13 and EZH2 were low in cisplatin- and paclitaxel-sensitive lung adenocarcinoma patients. Patients 4-6: HOXB13 and EZH2 were high in cisplatin- and paclitaxel-resistant lung adenocarcinoma patients. (F) Quantification for the levels of HOXB13 and EZH2 in cisplatin- and paclitaxel-sensitive (n=6) or resistant (n=9) lung adenocarcinoma patients (Unpaired Student's t -test, ** p<0.01).

Article Snippet: Briefly, deparaffinization and hydration were performed followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min, and then microwaved for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min. HOXB13 antibody (Santa Cruz, SC-28333, USA) and EZH2 antibody (Cell Signaling Technology, 30233s, USA) were used at 2 μg/ml in all experiments, and incubated at 4 °C overnight followed by the PV-9000 2-step plus Poly-HRP anti-Mouse/Rabbit IgG Detection system (Zhong Shan Jin Qiao, China).

Techniques: Transfection, Western Blot, ChIP-sequencing, Binding Assay, Control, Negative Control, Luciferase, Construct, Plasmid Preparation, Immunohistochemical staining

Journal: Theranostics

Article Title: HOXB13 networking with ABCG1/EZH2/Slug mediates metastasis and confers resistance to cisplatin in lung adenocarcinoma patients

doi: 10.7150/thno.29463

Figure Lengend Snippet: Cisplatin induces expression of HOXB13. (A) HOXB13 and its target genes ABCG1and EZH2 were induced in cisplatin-resistant A549 cells (A549 DDP) at the protein (Upper) and transcriptional levels (Lower) determined by Western blot or qPCR analyses. All these drug resistance genes were significantly upregulated by cisplatin induction (**p<0.01). (B) HOXB13, EZH2, and ABCG1 were transiently induced in the presence of 5 μM or 10 μM cisplatin treatment at indicated time points in A549 and H1299 cells, as detected by Western blot analysis. (C) Quantification of the bands to show that cisplatin upregulates HOXB13 and its target protein expression. (Unpaired Student's t -test, *p<0.05, **p<0.01, ***p<0.001) (D) HOXB13 and EZH2 levels were detected in drug-sensitive and drug-resistant PDX samples with or without cisplatin treatment by IHC. Left were detected by HOXB13 antibody and right were detected by EZH2 antibody.

Article Snippet: Briefly, deparaffinization and hydration were performed followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min, and then microwaved for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min. HOXB13 antibody (Santa Cruz, SC-28333, USA) and EZH2 antibody (Cell Signaling Technology, 30233s, USA) were used at 2 μg/ml in all experiments, and incubated at 4 °C overnight followed by the PV-9000 2-step plus Poly-HRP anti-Mouse/Rabbit IgG Detection system (Zhong Shan Jin Qiao, China).

Techniques: Expressing, Western Blot

Journal: Theranostics

Article Title: HOXB13 networking with ABCG1/EZH2/Slug mediates metastasis and confers resistance to cisplatin in lung adenocarcinoma patients

doi: 10.7150/thno.29463

Figure Lengend Snippet: Combination use of HOXB13 with ABCG1 and EZH2 gives high precision in predicting lung adenocarcinoma patients' outcome. (A) Combination of HOXB13 with its target gene expressions to predict lung adenocarcinoma prognosis. (B) Working model: HOXB13 induced by cisplatin confers lung adenocarcinoma patients' drug resistance by direct targeting to the newly identified drug resistance gene ABCG1 and also known drug resistance gene EZH2. Further, HOXB13 mediates metastasis of lung adenocarcinoma patients by direct targeting to EZH2 and Slug. Combination of HOXB13, ABCG1, EZH2 presents a better strategy to predict outcome or resistance to chemotherapy in lung adenocarcinoma patients.

Article Snippet: Briefly, deparaffinization and hydration were performed followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min, and then microwaved for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min. HOXB13 antibody (Santa Cruz, SC-28333, USA) and EZH2 antibody (Cell Signaling Technology, 30233s, USA) were used at 2 μg/ml in all experiments, and incubated at 4 °C overnight followed by the PV-9000 2-step plus Poly-HRP anti-Mouse/Rabbit IgG Detection system (Zhong Shan Jin Qiao, China).

Techniques: