Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: ELK1-induced upregulation of long non-coding RNA MIR100HG predicts poor prognosis and promotes the progression of osteosarcoma by epigenetically silencing LATS1 and LATS2.

doi: 10.1016/j.biopha.2018.10.029

Figure Lengend Snippet: Fig. 5. MIR100HG epigenetically suppresses the expression of LATS1 and LATS2 in OS cells. A. The nuclear location of MIR100HG was determined in OS cells. B. The binding probability between MIR100HG and RNA binding proteins (SUZ12, EZH2, LSD1) was analyzed by RNA-protein interaction prediction (RF and SVM scores > 0.5). C. RIP assay validated the interaction between MIR100HG and EZH2 in OS cells. D. The mRNA levels of EZH2’s potential targets were examined in OS cells in response to MIR100HG knockdown. E. EZH2 was silenced in OS cells by transfecting with sh-MIR100HG. F. The mRNA levels of EZH2’s potential targets were examined in OS cells in response to silenced EZH2. G. ChIP assay was applied to analyze the regulatory mechanism among MIR100HG, EZH2 and LATS1/2. The results obviously showed that silenced MIR100HG inhibited the binding ability of EZH2 to LATS1/2 promoter region and mediated the demethylation of H3K27me3 in OS cells. *P < 0.05, **P < 0.01 vs control group.

Article Snippet: The negative control, normal IgG (Proteintech), and antibodies against EZH2, SUZ12, LSD1 (Cell Signaling Technology, Beverly, MA) were bond with magnetic beads and cultured with the cell extract in RIP buffer.

Techniques: Expressing, Binding Assay, RNA Binding Assay, Knockdown, Control

Journal: Life Science Alliance

Article Title: BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells

doi: 10.26508/lsa.201900534

Figure Lengend Snippet: (A) Gene set enrichment analysis of the RNA-seq data from wild-type (WT) and KO Bmal1 embryonic stem cells (ESCs). Sets of the ESC-enriched genes and the targets of the pluripotency markers POU5F1, SOX2, and NANOG ( ; ) were used. (B) Grid highlighting the transcription factors (TFs) whose depletion leads to the more significant associated differential expression after their perturbation to Bmal1 KO. (C) Western blot of Polycomb proteins (EZH2 and SUZ12) in Bmal1 WT and KO ESCs. GAPDH was used as a loading control. (D) RNA expression of the indicated genes in Bmal1 WT and KO ESCs detected by RNA-seq. Bars represent mean ± SD. Two-tailed unpaired t test was performed. FPKM, fragments per million mapped reads; ns, not significant.

Article Snippet: For Western blotting, the following antibodies and dilutions were used: αBMAL1 (1:2,000, ab93806; Abcam), αPOU5F1 (1:4,000, sc-5279; Santa Cruz Biotechnology), αNANOG (1:2,000, A300-397A-2; Bethyl Laboratories), αZFP42 (1:1,000, sc-514643; Santa Cruz Biotechnology), αEZH2 (1:5,000, #5246S; Cell Signaling Technology), αSUZ12 (1:1,000, sc-271325; Santa Cruz Biotechnology), and α-ACTIN (1:2,000, sc-47778; Santa Cruz Biotechnology) or β-TUBULIN (1:2,500, sc-55529; Santa Cruz Biotechnology) as loading controls.

Techniques: RNA Sequencing, Quantitative Proteomics, Western Blot, Control, RNA Expression, Two Tailed Test

Journal: iScience

Article Title: Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal

doi: 10.1016/j.isci.2023.107035

Figure Lengend Snippet:

Article Snippet: Human Recombinant SUZ12 (NM_015355) Protein , Origene , TP302362.

Techniques: Control, Virus, Recombinant, Modification, Reverse Transcription, SYBR Green Assay, Cell Culture, Gel Extraction, Plasmid Preparation, Software

Journal: Development (Cambridge, England)

Article Title: Geminin deletion increases the number of fetal hematopoietic stem cells by affecting the expression of key transcription factors.

doi: 10.1242/dev.109454

Figure Lengend Snippet: Fig. 6. Ablation of geminin modulates epigenetic marks and SUZ12 binding on the HOXA9 gene locus. ChIP in human K562 cells transfected with either siLuc or siGmnn, using antibodies recognizing (A) RNA polymerase II, (B) H3K4me3, (C) H3K9me3 and (D) H3K27me3. Chromatin isolated from K562 cells was incubated with a geminin-specific polyclonal antiserum and anti-Suz12 and a tilling approach encompassing −4.0 kb to +0.5 kb on the HOXA9 genomic locus was used. (E) Enrichment for geminin at distal loci 4.0 and 3.5 kb upstream of the HOXA9 TSS. (F) Immunoprecipitation using an anti-Suz12 antiserum and chromatin from K562 cells stably transfected with either an shControl vector or shGeminin showed that SUZ12 was enriched in the same distal elements as geminin, whereas geminin knockdown almost eliminated SUZ12 binding. Enrichment was determined as fold change over non-specific IgG anti-log values. Results shown are representative of three independent experiments performed in triplicate; error bars indicate s.e.m.

Article Snippet: Monoclonal antibodies against Suz12 [mouse monoclonal; 05-1317 (clone 2AO9), Millipore] at 2 mg per 25 μg chromatin isolated from K562 cells stably expressing shRNAs (control or geminin; Origene) were used according to the manufacturer’s instructions.

Techniques: Binding Assay, Transfection, Isolation, Incubation, Immunoprecipitation, Stable Transfection, Plasmid Preparation, Knockdown

Journal: bioRxiv

Article Title: Polycomb group proteins protect latent Kaposi sarcoma-associated herpesvirus from episome clearance and HUSH-dependent chromatin silencing

doi: 10.1101/2025.02.10.637455

Figure Lengend Snippet: A) ChIP-qPCR analysis of KSHV infected HAP1 cell lines (48h p.i.) Investigated mark or factor is shown on top of each graph. Human control loci (hs) serve as a reference. (Mean + range; n= two independent ChIP replicates.) B) Read density coverage tracks of histone marks on KSHV, determined by ChIP-seq. C) Immunoblot analysis of HAP1 knockout clonal cell lines. Individual or combinatorial deletion of genes is indicated at the top of each blot. KDM2B band deleted in KDM2B KO cells is indicated with an asterisk symbol (*). D) Genome browser view of the locus containing KDM2B in WT , KDM2B KO and SUZ12 KO /KDM2B KO HAP1 cells, showing complete removal of the locus. E) Quantification of LANA IF (48 h p.i.) of dilution series of de novo KSHV infection of 4×10 4 SLK and HAP1 cells with indicated number of infectious particles (Mean + range; n= 3 independent infections).

Article Snippet: SUZ12 rescue experiments were performed by cloning human SUZ12 cDNA (Addgene # 24232) into a LeGO lentiviral vector ( Weber et al , 2008 ).

Techniques: Infection, Control, ChIP-sequencing, Western Blot, Knock-Out

Journal: bioRxiv

Article Title: Polycomb group proteins protect latent Kaposi sarcoma-associated herpesvirus from episome clearance and HUSH-dependent chromatin silencing

doi: 10.1101/2025.02.10.637455

Figure Lengend Snippet: A) ChIP-qPCR analysis of KSHV infected HAP1 cell lines (48h p.i.) Investigated mark or factor is shown on top of each graph. Human control loci (hs) serve as a reference. (Mean + range; n= two independent ChIP replicates.) B) Immunoblotting of SUZ12 recue cells C) EZH2 Inhibitor treatment (5 days, 10 µM EPZ-6438) of HAP1 cells shows loss of H3K27me3. D) ChIP-qPCR analysis of KSHV infected HAP1 cells (48h p.i.) after DMSO (WT-DMSO), 10 µM EPZ-6438 treatment (WT-EPZ-6438), SUZ12 KO , or Rescue HAP1 cells (SUZ12 KO + SUZ12). Investigated mark is shown on top of each graph. Human control loci (hs) serve as a reference. (Mean + range; n= two independent ChIP replicates.) E) Differential expression of human transcriptome of HAP1 cells DMSO or EPZ6438 treated as well as different PRC knockout cell lines 48h p.i.. Number of significantly changed genes is shown below. (UP: log2FoldChange >= 1, padj < 10e-6; DOWN: log2FoldChange <= -1, padj < 10e-6).

Article Snippet: SUZ12 rescue experiments were performed by cloning human SUZ12 cDNA (Addgene # 24232) into a LeGO lentiviral vector ( Weber et al , 2008 ).

Techniques: Infection, Control, Western Blot, Expressing, Knock-Out

Journal: bioRxiv

Article Title: Polycomb group proteins protect latent Kaposi sarcoma-associated herpesvirus from episome clearance and HUSH-dependent chromatin silencing

doi: 10.1101/2025.02.10.637455

Figure Lengend Snippet: A) KSHV episome numbers quantified by quantitative real-time PCR (qPCR) after KSHV de novo infection of HAP1 cell lines, shown as episomes normalized to day 2 p.i. B) KSHV-BAC16 and KSHV-BAC16-ΔORF50 C) infected cells were selected to achieve 100 % KSHV+ cultures. After removal of hygromycine B following episome quantifications were normalized to Day 0. D) KSHV episome numbers quantified by qPCR after KSHV de novo infection of HAP1 cells. HAP1 WT cells were treated with DMSO or 10 µM EPZ-6438 continuously during and after de novo infection. SUZ12 KO or Rescue HAP1 cells (SUZ12 KO + SUZ12) were infected in parallel with KSHV to compare the dynamic range of EZH2 inhibition affecting episomal maintenance. E) KSHV episome numbers quantified by qPCR after KSHV de novo infection of HAP1 cells. HAP1 WT cells were treated with DMSO or 10 µM EPZ-6438 continuously during and after de novo infection (EPZ-6438) or inhibitor treatment was initiated after 72h post infection (3d p.i. EPZ-6438) F) KSHV episome numbers quantified by qPCR after KSHV de novo infection of SLK cells. SLK cells were treated with DMSO or 10 µM EPZ-6438 continuously during and after de novo infection. G) KSHV episome numbers quantified by qPCR after KSHV de novo infection of HEK-293 cells. HEK-293 cells were treated with DMSO or 10 µM EPZ-6438 continuously during and after de novo infection. H and I) KSHV episome numbers quantified by qPCR in KSHV long-term infected SLK-1 cells (H) and SLKp cells (I). SLK-1 and SLKp cells were continuously treated with DMSO or 10 µM EPZ-6438. Slope of exponential regression (k) is shown next to each sample. Significance (sig.) of difference in slope between control (WT or DMSO) versus treatment (knockout or EPZ-6438) is shown next to each slope (k). P > 0.05 (n.s), P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***), P ≤ 0.0001 (****). Cell doublings in 3B and C were calculated according to growth rates depicted in

Article Snippet: SUZ12 rescue experiments were performed by cloning human SUZ12 cDNA (Addgene # 24232) into a LeGO lentiviral vector ( Weber et al , 2008 ).

Techniques: Real-time Polymerase Chain Reaction, Infection, Inhibition, Control, Knock-Out

Journal: Journal of Leukocyte Biology

Article Title: SUZ12 participates in the proliferation of PNH clones by regulating histone H3K27me3 levels

doi: 10.1002/jlb.2a1021-564r

Figure Lengend Snippet: FIGURE 2 mRNA expression levels and clinical correlation analysis. (A) Heat map of different gene expression levels in the same patient/HC; (B) MUC4 in PNH versus HC (p = 0.0025**); (C) SUZ12 in PNH versus HC (p = 0.0003***); (D) CPS1 in PNH versus HC (p = 0.0351*); (E) MSH6 in PNH versus HC; (F) NCOR2 in PNH versus HC; (G) clinical correlation analysis of SUZ12 expression level and Ret% and the proportion of PNH clones. (*p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: After fixation and permeabilization using a BD Cytofix/CytopermTM Fixation/Permeablization Kit (554714), PE rabbit anti-human SUZ12 antibody (CST, 73123) was used for labeling, and cellsweredetectedusing aBECKMANCOULTER instrument.

Techniques: Expressing, Gene Expression, Clone Assay

Journal: Journal of Leukocyte Biology

Article Title: SUZ12 participates in the proliferation of PNH clones by regulating histone H3K27me3 levels

doi: 10.1002/jlb.2a1021-564r

Figure Lengend Snippet: FIGURE 3 SUZ12 expression level and H3K27me3 level. (A) relative SUZ12 mRNA expression in CD59+ or CD59– peripheral blood leukocytes from PNH patients and peripheral blood leukocytes from healthy controls (HCs); (B) relative SUZ12 mRNA expression in HCs and PNH patients with or without mutations; (C) SUZ12 protein expression levels and H3K27me3 methylation levels in CD59+ or CD59– peripheral blood leukocytes from PNH patients and peripheral blood leukocytes from HCs (*p < 0.05, **p < 0.01, ***p < 0.001)

Article Snippet: After fixation and permeabilization using a BD Cytofix/CytopermTM Fixation/Permeablization Kit (554714), PE rabbit anti-human SUZ12 antibody (CST, 73123) was used for labeling, and cellsweredetectedusing aBECKMANCOULTER instrument.

Techniques: Expressing, Methylation

Journal: Journal of Leukocyte Biology

Article Title: SUZ12 participates in the proliferation of PNH clones by regulating histone H3K27me3 levels

doi: 10.1002/jlb.2a1021-564r

Figure Lengend Snippet: FIGURE 4 (A) SUZ12 expression levels and H3K27me3 levels in THP-1 WT and THP-1 KD cells; (B) relative expression level of SUZ12 mRNA after lentiviral transfection; (C) SUZ12 expression level in the nucleus after lentiviral transfection; (D) flow cytometry detection of SUZ12 expression in the nucleus after lentiviral transfection; (E) SUZ12 protein expression and H3K27me3 levels after lentiviral transfection (*p < 0.05)

Article Snippet: After fixation and permeabilization using a BD Cytofix/CytopermTM Fixation/Permeablization Kit (554714), PE rabbit anti-human SUZ12 antibody (CST, 73123) was used for labeling, and cellsweredetectedusing aBECKMANCOULTER instrument.

Techniques: Expressing, Transfection, Flow Cytometry