Journal: Cell Cycle

Article Title: Activation of HIV-1 LTR by Rad51 in microglial cells

doi: 10.4161/cc.9.18.12930

Figure Lengend Snippet: Expression of Rad51 in HIV-1 infected primary human microglial cells. (A) Quantitation of the changes in Rad51 levels was performed by densitometric analysis of the scanned X-ray films. The results were normalized relative to expression of Grb2 and are presented as a histogram. (B) p24 levels in the supernatant of the infected microglial cells during the course of HIV-1 infection as measured by ELISA and is shown over time in days post-infection.

Article Snippet: Rad51 siRNA was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Expressing, Infection, Quantitation Assay, Enzyme-linked Immunosorbent Assay

Journal: Cell Cycle

Article Title: Activation of HIV-1 LTR by Rad51 in microglial cells

doi: 10.4161/cc.9.18.12930

Figure Lengend Snippet: Effect of Rad51 or siRNA-Rad51 or p65 on HIV-1 LTR activity. (A) Schematic representation of the structural organization of the HIV-1 LTR enhancer region and basal/core promoter and the positions of the deletion mutants that were used in this study. (B) Primary human microglial cells were transfected with 100 ng of the HIV-1 LTR deletion mutants (−167/+66, −94/+66, −80/+66, −40/+66) fused to the luciferase reporter gene either alone or in combination with 300 ng of plasmids expressing Rad51 and/or p65 subunit of NFκB. The numbers represent the fold change in the HIV-1 promoter activity mediated by Rad51 and/or p65/NFκB. (C) The effect of silencing of p65 subunit of NFκB gene expression on HIV-1 LTR activity was assayed when by transfection the HIV-1 LTR plasmid (−167/+66) alone or in combination with 100 nM of p65 siRNA (sip65), non-targeting siRNA (ntsiRNA) or plasmid-expressing Rad51 with or without p65 siRNA. (E) Effect of silencing of Rad51 expression was also assayed by transfection of the HIV-1 LTR with 100 nM of Rad51 siRNA (siRad51), non-targeting siRNA (ntsiRNA) or plasmid-expressing p65/NFκB with or without Rad51 siRNA. The amounts of DNA in each transfection mixture were normalized with pcDNA. Luciferase activity was determined 48 hours after transfection. (D and E) The levels of expression of p65/NFκB and Rad51 were analyzed by Western blots for the lysates from the experiments shown in (C and E), respectively. Grb2 served as a loading control.

Article Snippet: Rad51 siRNA was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Activity Assay, Transfection, Luciferase, Expressing, Gene Expression, Plasmid Preparation, Western Blot, Control

Journal: Cell Cycle

Article Title: Activation of HIV-1 LTR by Rad51 in microglial cells

doi: 10.4161/cc.9.18.12930

Figure Lengend Snippet: Colocalization of Rad51 and p65/NFκB. Microglia were transfected with plasmid expressing p65/NFκB together with plasmids expressing either GFP-Rad51 or GFP. Cells transfected with GFP-Rad51 show bright green staining that is cytoplasmic, predominantly perinuclear and punctuate staining in the nucleus (D). Green staining in GFP-transfected control cells was uniformly distributed in nuclei and cytoplasm (A). By immunofluorescence we detected p65/NFκB protein in both nuclei and cytoplasm of infected cells (B and E rhodamine). Superimposition of both fluorochromes shows colocalization of both proteins in the perinuclear region and in the nuclei of cells (G). DAPI was used for labeling nuclear DNA (C and F).

Article Snippet: Rad51 siRNA was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, Control, Immunofluorescence, Infection, Labeling

Journal: Cell Cycle

Article Title: Activation of HIV-1 LTR by Rad51 in microglial cells

doi: 10.4161/cc.9.18.12930

Figure Lengend Snippet: Physical interaction between Rad51 and p65/NFκB. (A) Interaction between p65/NFκB and Rad51 was evaluated by immunoprecipitation (IP)/western blot analysis. Total cell lysate prepared from U-87MG cells was immunoprecipitated using anti-Rad51 antibody (lane 2). Nonimmune mouse serum (NMS) was used for the negative control for the IP reaction (lane 1). After IP, protein complexes were washed, separated by SDS-PAGE followed by Western blot analysis using anti-p65 antibody. Fifty micrograms of the total cell lysate protein extract was included on the gel as a positive control (lane 3). (B) Protein extracts from U-87MG cells were incubated with GST-Rad51 or its deletion mutants (1 µM) in a GST pull-down assay for identification of the region of Rad51 that binds to p65/NFκB. GST was used as a negative control for the binding reaction (lane 1). NFκB/p65 was assessed by Western blot. Fifty micrograms of total cell protein extract was used as a positive control for protein expression (lane 7). (C) The integrity of GST and the GST-Rad51 fusion proteins used in the assay was demonstrated directly by SDS-PAGE followed by Coomassie blue staining. (D) Schematic representation of the organization of Rad51, the deletion mutants and their relative binding to p65/NFκB.

Article Snippet: Rad51 siRNA was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Immunoprecipitation, Western Blot, Negative Control, SDS Page, Positive Control, Incubation, Pull Down Assay, Binding Assay, Expressing, Staining

Journal: Cell Cycle

Article Title: Activation of HIV-1 LTR by Rad51 in microglial cells

doi: 10.4161/cc.9.18.12930

Figure Lengend Snippet: Effect of Rad51 on the interaction of p65/NFκB with κB DNA sequence element of HIV-1 LTR measured by gel shift in nuclear extracts from U87 MG cells. (A) Approximately 100,000 cpm of synthetic [γ32P]-labeled double-stranded DNA oligonucleotide probe corresponding to the HIV-1 LTR κB site was incubated with 10 µg of nuclear extracts prepared from U87 MG cells transfected with 100 nmol siRNA for Rad51 (lane 3) or 200 nmol siRad51 (lane 4), 200 nmol non-target siRNA (lane 5), pcDNA6A-RAD51 (1–339) (lane 6), with p65/NFκB (lanes 11–14) and control pcDNA (lane 2). Labeled probe was also incubated with nuclear extracts prepared from pcDNA-transfected U87 MG cells in the presence of a specific DNA competitor (cold probe, lane 7), non-specific competitor (an unrelated dsDNA) (lanes 8 and 12), anti-p65 antibody (lanes 9 and 13) and normal mouse serum (NMS) (lanes 10 and 14). (B) Western blots were performed to determine the levels of expression of Rad51 with Lamin A as a loading control (lower part). (C) To identify the region of Rad51 involved in activation of the HIV-1 LTR, primary human microglial cells were transfected with 100 ng of LTR-luciferase plasmid (−167/+66) either alone or in combination with 300 ng of plasmids expressing Rad51: full length (1–339), Rad51 (1–160), Rad51 (1–80), Rad51 (1–40) and Rad51 (80–339). The amount of DNA in each transfection mixture was normalized with pCDNA6A. Luciferase activity was determined 48 hours after transfection. (D) Expression of Rad51 and deletion mutants was verified by western blot analysis of cell lysates prepared from the transfected primary human microglial cells using antibody against MYC-tagged Rad51.

Article Snippet: Rad51 siRNA was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Sequencing, Gel Shift, Labeling, Incubation, Transfection, Control, Western Blot, Expressing, Activation Assay, Luciferase, Plasmid Preparation, Activity Assay

Journal: Nature Communications

Article Title: Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia

doi: 10.1038/s41467-019-12960-6

Figure Lengend Snippet: EZH1 promotes AML1-ETO leukemia. a qPCR for EZH1 mRNA expression in leukemia cell lines ( n = 3). b Western blotting for EZH1 and EZH2 protein expression in leukemia cells lines and healthy human donors (HD). c qPCR for EZH1 mRNA expression in AML1-ETO-positive ( n = 62) and negative ( n = 60) leukemia patients. Median values are depicted by the horizontal lines. * P < 0.05. d Normalized EZH1 expression in primary cells of de novo AML patients (GSE6891) ( n = 347). NN refers to normal karyotype. Patient characteristics are summarized in Supplementary Table . e Correlation analysis for AML1-ETO and EZH1 mRNA expression in AML1-ETO-positive leukemia patients ( n = 62). R: Pearson correlation coefficients; R : indicates “the goodness of fit”. Statistical significance was calculated by Pearson correlation coefficients. f The association of EZH1 expression with overall survival (OS) and event free survival (EFS) in AML1-ETO-positive patients ( n = 62; from Fig. 1c) analyzed by the Kaplan–Meier estimate ( n = 62). g , h EZH1-depleted and control SKNO-1 cells were injected subcutaneously into the flanks of nude mice. The measurement of xenograft tumor size ( g ), and the visual analysis and tumor weight ( h ) ( n = 6 tumors/group). Data are expressed as mean values ± S.D. * P < 0.05, ** P < 0.01. i Representative images of H&E-stained and IHC-stained tumor sections (original magnification ×200). Scale bar, 100 µm. Insets show high-magnification images of the boxed areas. j – p Kasumi-1 and SKNO-1 cells (1–3 × 10 6 ) infected with EZH1 viruses were injected into NOD/SCID/γ c null immunodeficient mice through the tail-vein ( n = 5 mice/group). WBC analysis of peripheral blood (PB) ( j , k ), blast cells percentage in PB ( l ), Wright/Giemsa staining of bone marrow ( m ), graph illustration of the spleen weights ( n ), representative external views of the spleens ( o ) and H&E staining of sections of spleens, livers and lungs ( p ). Scale bars represent 100 µm (blue). Fig. a , data are expressed as mean values ± S.E.M. of triplicate samples from three independent experiments; Fig. n , data are expressed as mean values ± S.D. * P < 0.05, ** P < 0.01; Fig. c , Mann–Whitney U -tests; Figs. d , g , h , i , n , one-way ANOVA; Fig. f , Log-rank test; For box-whisker plots, box edges correspond to 25th and 75th percentiles, lines inside the box correspond to 50th percentiles, and whiskers include extreme data points. AE AML1-ETO. The data in Fig. b are representative of 3 independent experiments. Source data are included in the Source Data file

Article Snippet: The following expression plasmids were purchased from Addgene (Cambridge, USA): pCMV-AML1-ETO (ID 12428), HA-AE-W (wild type) (ID 12430), pSMP-EZH1_2 (ID 36360), pSMP-EZH1_1 (ID 36359), pSMP-EZH1_3 (ID 36361), HA-AE9a (ID 12433), pCMV5-AML1B (ID 12426), FLAG-ETO-W (wild type) (ID 12507), pMSCV-FLT3-ITD (ID 74499); from OriGene (Rockville, USA): FLAG-EZH1-W (wild type) (Myc-DDK-tagged)-Human enhancer of zeste homolog 1 (Drosophila; EZH1; ID RC202367) ; The scrambled and shRNA vectors were from BMGC RNAi: V2LMM_102131-ep300 , V3LHS_331296-ep300 , V3LHS_331297-ep300 , V2LHS_151492-EZH1 , V2LHS_151493-EZH1 , V2LHS_151495-EZH1 , TRCN0000002439 − EZH1 , TRCN000000 2440-EZH1 , TRCN0000002441-EZH1 , V2LHS_256397-SUZ12 , V2LHS_74301-SUZ12 , V2LHS_23172-EED , V2LHS_23174-EED .

Techniques: Expressing, Western Blot, Injection, Staining, Infection, MANN-WHITNEY, Whisker Assay

Journal: Nature Communications

Article Title: Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia

doi: 10.1038/s41467-019-12960-6

Figure Lengend Snippet: NHR1 domain of AML1-ETO interacts with the WD domain of EZH1. a Western blotting for anti-ETO and anti-EZH1 immunoprecipitates (lanes 3–6) from Kasumi-1 and SKNO-1 cells. IgG pull-down performed in parallel (lanes 7, 8) was used as a negative control. b Western blotting for anti-ETO immunoprecipitates from AML1-ETO-positive patient primary cells. IgG pull-down performed in parallel was used as a negative control. Pt, patient. c HEK293 cells were transfected for 48 h with HA-AE-W (wild type), FLAG-EZH1-W (wild type) alone or both. The anti-HA immunoprecipitates were subjected to Western blotting. d Upper: diagram of AML1-ETO domain constructs; lower: Western blotting for anti-His immunoprecipitates from HEK293 cells expressing His-AE-W (wild type), His-AE-no-NHR2, His-AE-no-NHR with FLAG-EZH1-W (wild type). e Upper: diagram of EZH1 domain constructs; lower: HEK293 cells were transfected for 48 h with HA-AE-W (wild type) and FLAG-EZH1-W (wild type), FLAG-EZH1-ΔSET, FLAG-EZH1-ΔSANT, or FLAG-EZH1-ΔWD. The anti-HA immunoprecipitates were subjected to Western blotting. f A model structure of the AML1-ETO/EZH1 complex. The EZH1 WD interaction domain and AML1-ETO NHR1 domain are shown in blue and magenta, respectively. A magnified view of the binding interface is shown in the inset, in which the potential interacting residues are represented by a ball-and-stick model. Bold letters indicate the critical amino residues for interactions proven by point mutations. g HEK293 cells were transfected for 48 h with HA-AE-W (wild type) and FLAG-EZH1-W (wild type) or the indicated mutant constructs. The anti-FLAG immunoprecipitates were subjected to Western blotting. IP Immunoprecipitation, WB Western blotting, AE AML1-ETO; The “Input” of each Fig is the immunoblot analysis for whole cell extracts showing the target protein levels. The data are representative of 3 independent experiments. Source data are included in the Source Data file

Article Snippet: The following expression plasmids were purchased from Addgene (Cambridge, USA): pCMV-AML1-ETO (ID 12428), HA-AE-W (wild type) (ID 12430), pSMP-EZH1_2 (ID 36360), pSMP-EZH1_1 (ID 36359), pSMP-EZH1_3 (ID 36361), HA-AE9a (ID 12433), pCMV5-AML1B (ID 12426), FLAG-ETO-W (wild type) (ID 12507), pMSCV-FLT3-ITD (ID 74499); from OriGene (Rockville, USA): FLAG-EZH1-W (wild type) (Myc-DDK-tagged)-Human enhancer of zeste homolog 1 (Drosophila; EZH1; ID RC202367) ; The scrambled and shRNA vectors were from BMGC RNAi: V2LMM_102131-ep300 , V3LHS_331296-ep300 , V3LHS_331297-ep300 , V2LHS_151492-EZH1 , V2LHS_151493-EZH1 , V2LHS_151495-EZH1 , TRCN0000002439 − EZH1 , TRCN000000 2440-EZH1 , TRCN0000002441-EZH1 , V2LHS_256397-SUZ12 , V2LHS_74301-SUZ12 , V2LHS_23172-EED , V2LHS_23174-EED .

Techniques: Western Blot, Negative Control, Transfection, Construct, Expressing, Binding Assay, Mutagenesis, Immunoprecipitation

Journal: Nature Communications

Article Title: Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia

doi: 10.1038/s41467-019-12960-6

Figure Lengend Snippet: EZH1 mediates AML1-ETO Lys43 methylation in vitro and in vivo. a Quantification of AML1-ETO lysine methylation ( n = 3). HEK293 cells were transfected with HA-AE-W (wild type) alone or plus FLAG-EZH1-W (wild type) for 48 h. The anti-HA immunoprecipitates were subjected to Western blotting. Data are expressed as mean values ± S.D. * P < 0.05. b Western blotting for anti-HA immunoprecipitates from HEK293 cells expressing HA-AE-W (wild type), FLAG-EZH2-W (wild type), or both. c Western blotting for anti-His immunoprecipitates from HEK293 cells expressing vehicle, His-AE-W (wild type) with FLAG-EZH1-W (wild type), or His-AE-W (wild type) with FLAG-EZH1-W (wild type) plus HA-EZH2-W (wild type). d Upper: diagram of AML1-ETO domain constructs; lower: Western blotting for anti-His immunoprecipitates from HEK293 cells expressing His-AE-W (wild type), His-AE-ΔNHR1, His-AE-ΔNHR2, or His-AE-ΔRUNT plus FLAG-EZH1-W (wild type). e Western blotting for anti-HA immunoprecipitates from HEK293 cells expressing either HA-AE9a, FLAG-EZH1-W (wild type) alone or both. f Western blotting for anti-HA immunoprecipitates from HEK293 cells expressing HA-AE-W (wild type), HA-AEK43R, or plus FLAG-EZH1-W (wild type). g Coomassie blue-stained gels showing the proteins used for in vitro methylation assays. h In vitro methylation assays. Upper: the purified GST-AE, GST-AE-K43R proteins were incubated with GST-EZH1-W (wild type) or GST-EZH1-ΔSET in the presence of SAM at 37 °C. Lower: the purified GST-AE proteins were incubated with GST-EZH1-W (wild type) or GST-EZH2-W (wild type) in the presence of SAM at 37 °C. The SAM labeled AE was measured by Epoch 2 microplate spectrophotometer. i HEK293 cells were transfected with HA-AE-W (wild type) plus His-EZH1-W (wild type) or His-EZH1-ΔSET for 48 h; anti-HA immunoprecipitates were subjected to Western blotting. * P < 0.05; Fig. a , one-way ANOVA; IP Immunoprecipitation, WB Western blotting, SAM S-adenosyl methionine, AE AML1-ETO. The “Input” of each Fig is the immunoblot analysis for whole cell extracts to show the target protein levels. meK43: our anti-AML1-ETO Lys43 antibody; meK: commercial lysine methylation antibody. The data are representative of 3 independent experiments. Source data are included in the Source Data file

Article Snippet: The following expression plasmids were purchased from Addgene (Cambridge, USA): pCMV-AML1-ETO (ID 12428), HA-AE-W (wild type) (ID 12430), pSMP-EZH1_2 (ID 36360), pSMP-EZH1_1 (ID 36359), pSMP-EZH1_3 (ID 36361), HA-AE9a (ID 12433), pCMV5-AML1B (ID 12426), FLAG-ETO-W (wild type) (ID 12507), pMSCV-FLT3-ITD (ID 74499); from OriGene (Rockville, USA): FLAG-EZH1-W (wild type) (Myc-DDK-tagged)-Human enhancer of zeste homolog 1 (Drosophila; EZH1; ID RC202367) ; The scrambled and shRNA vectors were from BMGC RNAi: V2LMM_102131-ep300 , V3LHS_331296-ep300 , V3LHS_331297-ep300 , V2LHS_151492-EZH1 , V2LHS_151493-EZH1 , V2LHS_151495-EZH1 , TRCN0000002439 − EZH1 , TRCN000000 2440-EZH1 , TRCN0000002441-EZH1 , V2LHS_256397-SUZ12 , V2LHS_74301-SUZ12 , V2LHS_23172-EED , V2LHS_23174-EED .

Techniques: Methylation, In Vitro, In Vivo, Transfection, Western Blot, Expressing, Construct, Staining, Purification, Incubation, Labeling, Spectrophotometry, Immunoprecipitation

Journal: Nature Communications

Article Title: Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia

doi: 10.1038/s41467-019-12960-6

Figure Lengend Snippet: AML1-ETO Lys43 methylation is essential for AML1-ETO transcriptional activity. a Venn diagram illustrating the number of genes in which the peaks of AML1-ETO co-localize with those of EZH1, p300, meK43, or aceK43 in Kasumi-1 cells determined by anti-AML1-ETO (specific to AE, but not wild-type ETO or AML1), anti-EZH1, anti-p300, anti-meK43 (methylated AEK43, our developed antibody) or anti-aceK43 (acetylated AEK43, our developed antibody). b UCSC Genome Browser image depicting the human methylated K43-AML1-ETO and EZH1 loci demonstrating a co-localization of peaks at DIP2B promoter. c ChIP-qPCR showing the co-occupancy of AML1-ETO, EZH1, me-K43 at AML1-ETO target promoters in Kasumi-1 cells using an AML1-ETO specific antibody, anti-EZH1 or anti-me-K43 ( n = 3). d , e qPCR for changes in the AML1-ETO and EZH1 target gene expression in Kasumi-1 cells with knockdown of AE ( d ) or EZH1 ( e ) ( n = 3). f , g qPCR for changes in AML1-ETO and EZH1 target gene expression in HEK293 cells transfected with AE-W (wild type), AE-ΔNHR1 or AEK43R ( n = 3). h qPCR for changes in AML1-ETO and EZH1 target gene expression in Kasumi-1 cells transfected with the indicated mutant constructs ( n = 3). i ChIP-qPCR assessing the enrichment of AE and AEK43R in the target promoters ( n = 3). * P < 0.05; Fig. i , one-way ANOVA; AE AML1-ETO, meK43 methylated AEK43, aceK43 acetylated AEK43. The data are representative of 3 independent experiments. Data are expressed as mean values ± S.D. Source data are included in the Source Data file

Article Snippet: The following expression plasmids were purchased from Addgene (Cambridge, USA): pCMV-AML1-ETO (ID 12428), HA-AE-W (wild type) (ID 12430), pSMP-EZH1_2 (ID 36360), pSMP-EZH1_1 (ID 36359), pSMP-EZH1_3 (ID 36361), HA-AE9a (ID 12433), pCMV5-AML1B (ID 12426), FLAG-ETO-W (wild type) (ID 12507), pMSCV-FLT3-ITD (ID 74499); from OriGene (Rockville, USA): FLAG-EZH1-W (wild type) (Myc-DDK-tagged)-Human enhancer of zeste homolog 1 (Drosophila; EZH1; ID RC202367) ; The scrambled and shRNA vectors were from BMGC RNAi: V2LMM_102131-ep300 , V3LHS_331296-ep300 , V3LHS_331297-ep300 , V2LHS_151492-EZH1 , V2LHS_151493-EZH1 , V2LHS_151495-EZH1 , TRCN0000002439 − EZH1 , TRCN000000 2440-EZH1 , TRCN0000002441-EZH1 , V2LHS_256397-SUZ12 , V2LHS_74301-SUZ12 , V2LHS_23172-EED , V2LHS_23174-EED .

Techniques: Methylation, Activity Assay, Expressing, Transfection, Mutagenesis, Construct

Journal: Nature Communications

Article Title: Protein lysine 43 methylation by EZH1 promotes AML1-ETO transcriptional repression in leukemia

doi: 10.1038/s41467-019-12960-6

Figure Lengend Snippet: EZH1-mediated Lys43 methylation is essential for leukemic cell expansion. a Colony-forming assays in C1498 cells transfected with HA-AE-W (wild type) and FLAG-EZH1-W or the indicated mutant constructs ( n = 3). b Graph illustrates the average of the spleen weights from leukemic mice ( n = 3 mice/group) engrafted with C1498-transfected cells (0.1 × 10 6 , from a ). c Wright/Giemsa staining of BM and H&E staining of sections from the spleens, lungs, and livers of leukemic mice engrafted with the transfected C1498 cells (from a ). Scale bars represent 50 µm (red) and 100 µm (blue). d Kaplan–Meier survival curves of the engrafted mice. The mice transplanted with bone marrow cells co-expressing AML1-ETO plus FLT3-ITD developed fatal leukemia ( n = 5). e BM cells transduced with AML1-ETO plus FLT3-ITD or AML1-ETOK43R plus FLT3-ITD viruses were injected into C57BL/6 J mice ( n = 5 mice/group) through the tail vein. FACS analysis of the engrafted recipient BM cells from the representative disease mice. f , g Blast cell percentage by immunophenotype analysis ( f ) and morphological analysis ( g ) of hematopoietic cells in bone marrow from the representative disease mice (from d and e ). Scale bars represent 50 µm (red). h Graph illustrates the average of the spleen weights (from d and e ) ( n = 5). i Representative external views of the spleens from the leukemic mice (from d and e ). j H&E staining of sections from the spleens, lungs, and livers of the leukemic mice (from d and e ). Note, Fig. a , data are expressed as mean values ± S.E.M. of triplicate samples from three independent experiments; Fig. b , h , graphs are the average of spleen weight and data are expressed as mean values ± S.D; Fig. j , original magnification ×200 and scale bars represent 100 µm (blue); BM bone marrow, AE AML1-ETO; * P < 0.05; Fig. a , b , f , h , one-way ANOVA; d , Log-rank test; For box-whisker plots, box edges correspond to 25th and 75th percentiles, lines inside the box correspond to 50th percentiles, and whiskers include extreme data points. Source data are provided as a Source Data file

Article Snippet: The following expression plasmids were purchased from Addgene (Cambridge, USA): pCMV-AML1-ETO (ID 12428), HA-AE-W (wild type) (ID 12430), pSMP-EZH1_2 (ID 36360), pSMP-EZH1_1 (ID 36359), pSMP-EZH1_3 (ID 36361), HA-AE9a (ID 12433), pCMV5-AML1B (ID 12426), FLAG-ETO-W (wild type) (ID 12507), pMSCV-FLT3-ITD (ID 74499); from OriGene (Rockville, USA): FLAG-EZH1-W (wild type) (Myc-DDK-tagged)-Human enhancer of zeste homolog 1 (Drosophila; EZH1; ID RC202367) ; The scrambled and shRNA vectors were from BMGC RNAi: V2LMM_102131-ep300 , V3LHS_331296-ep300 , V3LHS_331297-ep300 , V2LHS_151492-EZH1 , V2LHS_151493-EZH1 , V2LHS_151495-EZH1 , TRCN0000002439 − EZH1 , TRCN000000 2440-EZH1 , TRCN0000002441-EZH1 , V2LHS_256397-SUZ12 , V2LHS_74301-SUZ12 , V2LHS_23172-EED , V2LHS_23174-EED .

Techniques: Methylation, Transfection, Mutagenesis, Construct, Staining, Expressing, Transduction, Injection, Whisker Assay