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: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of endogenous EZH2 (A) or SUZ12 (B) immunostaining in MDA-MB-231 and BoM-1833 cells. Insets highlight exemplary nuclear bodies of EZH2 or SUZ12 accumulation (arrows) in the BoM-1833 cells. Scale bar: 10 µm. Images were acquired and are displayed with identical settings. ( C ) Violin plot quantifying PRC2 body diameter in BoM-1833 cells. Each dot represents a single PRC2 body; data from 3 biological replicates (N = 16–32 cells). ( D ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in A-B. Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.0102. Error bars indicate mean ±SEM. ( E ) Representative confocal fluorescence microscopy image of BoM-833 cells stained for endogenous PRC2 (SUZ12, green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PRC2 body. Scale bar: 5 µm. ( F ) Schematic representation of the 3D photo-biotinylation approach used to map the proteome of endogenous PRC2 bodies. Total EZH2 (green) is spatially distributed within the cell and selectively photo-biotinylated at defined regions of interest (magenta) upon light activation. Following cell lysis, biotinylated proteins are captured using avidin-based immunoprecipitation and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The figure was created using Biorender. ( G ) Volcano plot illustrating the proteomic content of PRC2 bodies in BoM-1833 cells. Analysis was performed on the 1384 proteins identified as enriched in the labeled versus control condition in all 4 biological repeats, with unique peptides ≥ 2, fold change ≥ 1.5; and t-test significance ≤ 0.05. The x-axis represents the log 2 enrichment ratio (2P/CTL), and the y-axis represents the -log 10 p-value, indicating statistical significance. The dotted horizontal line corresponds to the p-value threshold (p < 0.05). Members of the core PRC2 complex are labeled in green. ( H ) Representative confocal fluorescence microscopy images of endogenous PHF19 immunostaining in MDA-MB-231 and BoM-1833 cells. The arrow highlights exemplary accumulations of PHF19 within nuclear bodies in BoM-1833 cells. Scale bar: 20 µm. The images were acquired and are displayed with identical settings. ( I ) Violin plot showing the quantification of endogenous PHF19 body diameter in BoM-1833 cells based on the images representatively shown in (H). Data represent measurements from N = 14–17 cells across n = 3 biological replicates, with each dot representing the diameter of a single PHF19 body. Biological repeats are color coded. ( J ) Quantification of percentage of cell nuclei with PHF19 bodies in MDA-MB-231 and BoM-1833 cells, based on the images representatively shown in (I). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, p=0.003. Error bars indicate mean ±SEM. ( K ) Representative confocal fluorescence microscopy image of endogenous PHF19 (green) and H3K27me3 (magenta) immunostaining in BoM-1833 cells. The arrow indicates an exemplary area of co-localization at a PHF19 body. Scale bar: 5 µm. ( L ) Representative confocal fluorescence microscopy images of BoM-1833 cells, 24 h post transfection with a GFP-PHF19 (green) expression plasmid and immunostained for endogenous core PRC2 subunits (SUZ12, purple). The arrow indicates an exemplary area of co-localization. Scale bar: 10 µm.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Immunostaining, Staining, Activation Assay, Lysis, Avidin-Biotin Assay, Immunoprecipitation, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Labeling, Control, Transfection, Expressing, Plasmid Preparation

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A-B ) Representative confocal fluorescence microscopy images of BoM-1833 cells transfected with the indicated siRNAs. Cells were fixed 96 hours post-transfection and immunostained for endogenous EZH2 (A) or SUZ12 (B). Regions of interest (ROIs) are highlighted, with inset images showing magnified views of the immunostained cells. Scale bar: 10 µm. Images that are to be directly compared where imaged and are displayed with identical settings. ( C ) Quantification of the percentage of nuclei exhibiting PRC2 bodies in BoM-1833 cells treated as in (A-B) and immunostained for PRC2 core subunits. Data represent measurements from N = 50–60 cells across n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, *** = 0.0003, ns= not significant. Error bars indicate mean ±SD. ( D ) BoM-1833 cells were transfected with the indicated siRNAs and lysed 96 hours later for Western blot analysis using the specified antibodies. GAPDH was used as loading control. ( E-I ) Densitometric analysis of PHF19 (E), EZH2 (F), SUZ12 (G), PHF1 (H) and MTF2 (I) protein levels in cell lysates obtained from BoM-1833 cells treated as described in (D). GAPDH was used for relative normalization of the chemiluminescence signal obtained for the different PRC2 subunits. Data represent measurements from n = 3 biological replicates, whereby the values for siPHF19 are reported relative to the mean value of the control (siNT) within each biological replicate. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA testing, **** < 0.0001, ns = not significant. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Fluorescence, Microscopy, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: PHF19 drives PRC2 sub-nuclear compartmentalization to promote motility in TNBC cells

doi: 10.1101/2025.03.13.642950

Figure Lengend Snippet: ( A ) PHF19 gene expression analysis across a TCGA BRCA cohort sorted by molecular subtype subtype. Box plots display the expression levels of PHF19 in normal (grey) and tumor (green) tissue for the indicated breast cancer subtypes. Data are derived from TCGA/GTEx datasets and visualized using GEPIA2. Statistical significance between tumor and normal samples was determined by unpaired t-test (*p < 0.05). n= 291 (Normal), 194 (Luminal B), 415 (Luminal A), 66 (HER2), 135 (Basal-like). ( B-C ) Representative confocal microscopy images of EZH2 (B) and SUZ12 (C) immunostaining in the indicated cell lines. Scale bar: 20 µm. Images that are to be directly compared were recorded and are displayed using identical settings. ( D ) Quantification of the percentage of cell nuclei with PRC2 bodies in the indicated cell lines based on confocal microscopy images as shown in (B-C). Data represent measurements from N = 35– 55 cells across n = 3 biological replicates. Biological repeats are color coded. ( E ) Representative immunoblot analysis of full cell lysates prepared from the indicated cell lines and using the annotated antibodies. GAPDH was used as the loading control. ( F-G ) Densitometric quantification of EZH2, SUZ12 (F) and PCL family (G) subunit protein expression in the TNBC cell line panel used in this work. GAPDH was used for normalization of the chemiluminescence signal of the PRC2 subunits across cell lines. The data for siPHF19 are reported relative to the mean values for the siNT control. Data represent measurements from n = 3 biological replicates, error bars are mean ±SD. Measurements stemming from cell lines forming detectable PRC2 bodies by Airyscan microscopy were highlighted in red. ( H-I ) Representative confocal fluorescence microscopy images showing co-immunostaining of H3K27me3 with the endogenous PRC2 core subunit SUZ12 (H) and PHF19 (I) in MDA-MB-436 cells. Arrows indicate exemplary regions of colocalization. Scale bar: 10 µm (H), 5 µm (I). ( J ) Violin plot showing the quantification of PRC2 core and PHF19 protein body diameter as based on the images representatively shown in (F-G). Data represent measurements from N = 14–29 (core PRC2 subunits) and N= 19-22 (PHF19) cells across n = 3 biological replicates, with each dot representing the diameter of a single protein body. Biological repeats are color coded. ( K ) Representative confocal fluorescence microscopy images of MDA-MB-436 cells, 24 h post transfection with GFP-PHF19 (green) and immunostained for endogenous SUZ12 (purple). The arrow indicates an exemplary area of co-localization. Scale bar: 5 µm. ( L-M ) MDA-MB-436 cells were transfected with the indicated siRNAs followed by fixation 96 h later and immunostaining for endogenous EZH2 (L) or SUZ12 (M). The bottom row shows magnified views of the cropped fields of view. Images that are to be directly compared were acquired and are displayed using identical settings. Scale bar: 10 µm ( N ) Quantification of percentage of cell nuclei with PRC2 bodies in MDA-MB-436 cells transfected with the indicated siRNAs and imaged as representatively shown in (L-M). Data represent measurements from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via one-way ANOVA, ****= 0.001, ns= not significant. Error bars indicate mean ±SD. ( O ) MDA-MB-436 were treated as described in (L-M), followed by cell lysis. The material was analyzed by Western blot using the indicated antibodies. See also Figure S4. ( P , S ) Representative confocal microscopy images and ( R , T ) quantification of HS578T (P, R) and BT549 (S, T) fixed 24 h after transfection with a plasmid encoding for GFP-PHF19 (magenta) and immunostained for endogenous SUZ12 (PRC2 core). ROIs (Regions of Interest) are highlighted and magnified, showing the endogenous localization of SUZ12 in cells transfected with GFP-PHF19 (ROI 1) versus un-transfected cells (ROI 2). Scale bar: 20 µm. The bar diagrams show the endogenous SUZ12 localization phenotype in relation to the GFP-PHF19 expression status. Data represent measurements from N = 7–30 cells from n = 3 biological replicates. Biological repeats are color coded. Statistical significance was determined via unpaired t-test, * = 0.0123, **= 0.0038. Error bars indicate mean ±SD.

Article Snippet: The cells were then incubated with the rabbit anti-EZH2 antibody (5246, Cell signaling, USA) for 4 hours at RT, washed 3 times with PBST for 5 min and then incubated with Alexa Fluor™ 647 secondary antibody (A-21245, ThermoFisher, USA) for 2 hours.

Techniques: Gene Expression, Expressing, Derivative Assay, Confocal Microscopy, Immunostaining, Western Blot, Control, Microscopy, Fluorescence, Transfection, Lysis, Plasmid Preparation

Journal: Blood cancer journal

Article Title: Maternal embryonic leucine zipper kinase is a novel target for diffuse large B cell lymphoma and mantle cell lymphoma.

doi: 10.1038/s41408-019-0249-x

Figure Lengend Snippet: Fig. 3 OTSSP167 treatment affects lymphoma cell cycle progression. a Proliferating lymphoma cells were incubated for 24 h with OTSSP167 (6.25, 12.5, and 25 nM) and subsequently with BrdU for 3 h and analyzed using the BrdU proliferation assay kit. Results shown are mean ± SD of three independent experiments. *p < 0.05. b Cell cycle analysis was performed after 24 h of OTSSP167 treatment (12.5 and 25 nM) using PI staining. Results shown are mean ± SD of three independent experiments. *p < 0.05 and **p < 0.01. c Protein levels of (p)FOXM1, Cdc25B, Cyclin B1, Aurora A kinase (AurA), Plk-1, and EZH2 were determined in DLBCL and MCL cell lines after 24 h of 25 nM OTSSP167 treatment. β-actin was used as loading control. One experiment representative of 3 is shown. d Scheme of the most examined downstream targets of MELK inhibition in cancer. Green arrows indicate activation and red arrows indicate inhibition/suppression. There is still some controversy whether MELK activates or inhibits p53 (AUR B: Aurora B kinase, CENP A/B: centromere protein A/B).

Article Snippet: Antibodies were used against MELK (#2274), Aurora B kinase (#3094), cyclin B1 (#4138), pFOXM1 (#14170), EZH2 (#4905), Cdc25B (#9525), Plk-1 (#4535), Aurora A kinase (#3092), PARP (#9542), Mcl1 (#5453), pBcl-2 (#2827), Bcl-xL (#2764), caspase-3 (#9662) and β-actin (#4967) (all from Cell Signaling Technology, Leiden, the Netherlands) and FOXM1 (sc_376471), Bcl-2 (sc_492) and pBcl-xL (sc_101644) (Santa Cruz, Heidelberg, Germany).

Techniques: Incubation, Proliferation Assay, Cell Cycle Assay, Staining, Control, Inhibition, Activation Assay

Journal: Frontiers in cell and developmental biology

Article Title: EZH2-Inhibited MicroRNA-454-3p Promotes M2 Macrophage Polarization in Glioma.

doi: 10.3389/fcell.2020.574940

Figure Lengend Snippet: FIGURE 1 | M2 macrophage polarization in patients with glioma is associated with EZH2 overexpression. (A) Immunohistochemistry analysis of EZH2 in clinical samples of different grades of gliomas (×200). (B) Immunohistochemistry analysis of CD206 in clinical samples of different grades of gliomas (×200). (C) EZH2 and CD206 immunohistochemical scores of clinical specimens of different grades of gliomas. (D) Pearson correlation analysis of EZH2 and CD206 immunohistochemical scores in glioma clinical samples. (E) EZH2 expression in glioma clinical specimens determined by RT-qPCR. (F) The expression of IL-6 in glioma clinical specimens determined by RT-qPCR. (G) The expression of IL-8 in glioma clinical specimens determined by RT-qPCR. (H) The expression of MIP-3α in glioma clinical specimens determined by RT-qPCR. n = 30 in WHO II group; n = 30 in WHO III group; n = 30 in WHO IV group; n = 30 in normal group. *p < 0.05 vs. normal brain tissues. #p < 0.05 vs. WHO II glioma tissues. &p < 0.05 vs. WHO III glioma tissues. All measurement data were shown as mean ± standard deviation. Data between two groups were compared by unpaired t-test, while comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s post-hoc test. Pearson correlation analysis was performed to observe the correlation of indicators.

Article Snippet: The primary antibodies (Proteintech Group Inc.) to EZH2 (1: 500), CD206 (1: 500), PTEN (1: 500, 22034-1-AP), and YTHDF2 (1: 500, 24744-1-AP) were supplemented for slice incubation, followed by slice culture with horseradish peroxidase (HRP)tagged secondary antibodies.

Techniques: Over Expression, Immunohistochemistry, Immunohistochemical staining, Expressing, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in cell and developmental biology

Article Title: EZH2-Inhibited MicroRNA-454-3p Promotes M2 Macrophage Polarization in Glioma.

doi: 10.3389/fcell.2020.574940

Figure Lengend Snippet: FIGURE 2 | The growth of glioma and M2 macrophage polarization is repressed by silencing EZH2 in nude mice. (A) The successful establishment of glioma xenograft model in nude mice determined using HE staining (×200). (B) Western blots of EZH2 protein. (C) Western blot analysis to verify the silencing efficiency of Lenti-EZH2. (D) The expression of N-cadherin and Vimentin proteins measured by Western blots. (E) A172 cells treated with Lenti-EZH2 or Lenti-HK were co-injected with polarized macrophages into the axilla of nude mice, and the size of glioma was measured and analyzed 2 months later. (F) Tumor size of mice. (G) The GFP fluorescence intensity of glioma formation in nude mice measured by an in vivo imaging system (IVIS spectrum), and the signal intensity of ROI reflected the growth of the tumor. (H) Statistical analysis of ROI signal intensity. (I) Immunofluorescence detection of the expression of CD206, MIP-3α, IL-6, and IL-8 in tumor tissues of mice (×400). (J) Statistical analysis of immunofluorescence intensity of CD206, MIP-3α, IL-6, and IL-8 in mice. (K) The expression of MIP-3α, IL-6, and IL-8 in plasma of nude mice evaluated by ELISA. *p < 0.05 vs. nude mice injected with Lenti-HK-treated glioma cells and polarized macrophages. n = 8. All measurement data were shown as mean ± standard deviation. Data between two groups were compared by unpaired t-test, while comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s post-hoc test.

Article Snippet: The primary antibodies (Proteintech Group Inc.) to EZH2 (1: 500), CD206 (1: 500), PTEN (1: 500, 22034-1-AP), and YTHDF2 (1: 500, 24744-1-AP) were supplemented for slice incubation, followed by slice culture with horseradish peroxidase (HRP)tagged secondary antibodies.

Techniques: Staining, Western Blot, Expressing, Injection, In Vivo Imaging, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in cell and developmental biology

Article Title: EZH2-Inhibited MicroRNA-454-3p Promotes M2 Macrophage Polarization in Glioma.

doi: 10.3389/fcell.2020.574940

Figure Lengend Snippet: FIGURE 3 | Downregulated EZH2 alters the microenvironment of glioma and suppresses the polarization of M2 macrophages co-cultured in vitro. (A) The proportion of CD11b+ CD206+ cells in the co-cultured cells determined by flow cytometry. (B) The expression of IL-8, MIP-3α, and IL-6 in macrophages measured by RT-qPCR. (C) The expression of IL-8, IL-6, and MIP-3α in macrophages detected by immunofluorescence (×400). (D) Statistical analysis of the fluorescence intensity of IL-8, IL-6, and MIP-3α in macrophages. (E) The content of IL-8, MIP-3α, and IL-6 in cell culture supernatant detected by ELISA. (F) The expression of M1 polarization markers II1b, Cd86, and Nos2 as well as M2 markers Cd163, Ym1, and Mrc1 in macrophages determined by RT-qPCR. *p < 0.05 vs. treatment with Lenti-HK. All measurement data were shown as mean ± standard deviation. Data between two groups were compared by unpaired t-test. Experiments were repeated three times independently.

Article Snippet: The primary antibodies (Proteintech Group Inc.) to EZH2 (1: 500), CD206 (1: 500), PTEN (1: 500, 22034-1-AP), and YTHDF2 (1: 500, 24744-1-AP) were supplemented for slice incubation, followed by slice culture with horseradish peroxidase (HRP)tagged secondary antibodies.

Techniques: Cell Culture, In Vitro, Cytometry, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in cell and developmental biology

Article Title: EZH2-Inhibited MicroRNA-454-3p Promotes M2 Macrophage Polarization in Glioma.

doi: 10.3389/fcell.2020.574940

Figure Lengend Snippet: FIGURE 4 | EZH2 contributes to miR-454-3p downregulation via DNA methylation to promote polarization of M2 macrophages. (A) The expression of miR-454-3p in clinical samples of different grades of glioma detected by RT-PCR. *p < 0.05 vs. normal brain tissues, #p < 0.05 vs. WHO II glioma tissues, &p < 0.05 vs. WHO III glioma tissues. (B) The efficiency of miR-454-3p and miRZIP-454 verified by RT-qPCR. *p < 0.05 vs. the treatment with control-miR, #p < 0.05 vs. the treatment with control-miRZIP. (C) Pearson correlation analysis of EZH2 and miR-454-3p expression in glioma clinical samples. (D) RT-qPCR to detect the expression of miR-454-3p and PTEN in glioma cells after EZH2 inhibition. *p < 0.05 vs. the treatment with Lenti-HK. (E) The proportion of CD11b+ CD206+ cells in cells after co-culture of miR-454-3p-overexpressed/knockout A172 cells with THP-1 cells detected by flow cytometry. (F) The expression of IL-8, IL-6, and MIP-3α in the macrophages after co-culture with A172 cells treated with Lenti-PTEN determined by RT-qPCR. (G) Statistical analysis of the fluorescence intensity of IL-8, IL-6, and MIP-3α in the tumor microenvironment. (H) The levels of IL-8, IL-6, and MIP-3α in the supernatant detected by ELISA. p < 0.05 vs. the treatment with control-miR, #p < 0.05 vs. the treatment with control-miRZIP. (I) CpG island and primer prediction. (J) MethPrime analysis of promoter CpG islands of miR-454-3p. All measurement data were shown as mean ± standard deviation. Data between two groups were compared by unpaired t-test, while comparisons among multiple groups were performed using one-way ANOVA, followed by Tukey’s post-hoc test. Pearson correlation analysis was performed to observe the correlation of indicators. Experiments were repeated three times independently.

Article Snippet: The primary antibodies (Proteintech Group Inc.) to EZH2 (1: 500), CD206 (1: 500), PTEN (1: 500, 22034-1-AP), and YTHDF2 (1: 500, 24744-1-AP) were supplemented for slice incubation, followed by slice culture with horseradish peroxidase (HRP)tagged secondary antibodies.

Techniques: DNA Methylation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Control, Inhibition, Co-Culture Assay, Knock-Out, Cytometry, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in cell and developmental biology

Article Title: EZH2-Inhibited MicroRNA-454-3p Promotes M2 Macrophage Polarization in Glioma.

doi: 10.3389/fcell.2020.574940

Figure Lengend Snippet: FIGURE 7 | The scheme of the mechanism by which EZH2 affects glioma tumorigensis. EZH2 inhibits miR-454-3p to enhance the binding to m6A reading protein YTHDF2, whereby promoting m6A modification of PTEN and inducing M2 macrophage polarization in glioma and tumorigensis.

Article Snippet: The primary antibodies (Proteintech Group Inc.) to EZH2 (1: 500), CD206 (1: 500), PTEN (1: 500, 22034-1-AP), and YTHDF2 (1: 500, 24744-1-AP) were supplemented for slice incubation, followed by slice culture with horseradish peroxidase (HRP)tagged secondary antibodies.

Techniques: Binding Assay

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: 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:

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