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: The Journal of clinical endocrinology and metabolism

Article Title: Enhancer of zeste homolog 2 overexpression has a role in the development of anaplastic thyroid carcinomas.

doi: 10.1210/jc.2010-1784

Figure Lengend Snippet: FIG. 2. Immunohistochemical analysis of EZH2 expression in normal and neoplastic thyroid tissues. A, Normal thyroid (1). EZH2 staining was absent in follicular epithelial cells (magnification, 20). In papillary (2) and follicular (3) carcinoma, respectively, only few nuclei show a weak staining (magnification, 20) (4). In anaplastic carcinomas, a diffuse and intense staining is present (magnification, 20). B, Summary of the immunohistochemical data for EZH2 protein expression. Samples examined were as follows: six normal thyroids, eight PTC, seven FTC, and 12 ATC.

Article Snippet: The antibodies used were as follows: EZH2 (Cell signaling, Danvers, MA); SUZ12 and H3K27me3 (Upstate, Billerica, MA); EED and histone H3 lysine 3 trimethylation (H3K4me3) (Abcam, Cambridge, UK); glyceraldehyde-3-phosphate dehydrogenase, cyclin B1, and cadherin E (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Enhancer of zeste homolog 2 overexpression has a role in the development of anaplastic thyroid carcinomas.

doi: 10.1210/jc.2010-1784

Figure Lengend Snippet: FIG. 1. Analysis of enhancer of zeste homolog 2 (EZH2), embryonic ectoderm development (EED), and suppressor of zeste 12 homolog (SUZ12) expression in surgically removed human thyroid carcinomas of different histotypes. A and B, Quantitative RT-PCR analyses of EZH2, EED, and SUZ12 gene expression. The fold change indicates the relative change in expression levels between thyroid carcinomas and normal thyroid tissues, assuming that the mean value of three normal thyroid samples (NT) is equal to 1. The scale bars represent the mean SE (n 3). Results are reported as mean expression values of three independent experiments, with error bars indicating SE. The values represent statistically significant variations (with a P 0.05) between papillary, follicular, and anaplastic carcinomas and normal thyroid. C and D, Western blot analyses of EZH2, EED, and SUZ12 protein level. Blot against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been performed as control for equal protein loading. HeLa nuclear extract has been loaded as positive control for used antibodies; however, the EED protein expression was almost undetectable in HeLa cells.

Article Snippet: The antibodies used were as follows: EZH2 (Cell signaling, Danvers, MA); SUZ12 and H3K27me3 (Upstate, Billerica, MA); EED and histone H3 lysine 3 trimethylation (H3K4me3) (Abcam, Cambridge, UK); glyceraldehyde-3-phosphate dehydrogenase, cyclin B1, and cadherin E (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Western Blot, Control, Positive Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Enhancer of zeste homolog 2 overexpression has a role in the development of anaplastic thyroid carcinomas.

doi: 10.1210/jc.2010-1784

Figure Lengend Snippet: FIG. 3. Analysis of EZH2 expression in human thyroid carcinoma cell lines. A, Western blot analysis of EZH2 protein in normal and thyroid carcinoma cell lines. B, Semiquantitative (RT)-PCR analyses of EZH2 expression in 8505C, ACT-1, and FRO mass populations knocked down for EZH2 at mRNA level. Glucose-6-phosphate dehydrogenase (G6PDH) gene expression was evaluated as a control to normalize the amount of used RNAs. C, Western blot analysis of EZH2 protein level in 8505C, ACT-1, and FRO mass populations knocked down for EZH2.

Article Snippet: The antibodies used were as follows: EZH2 (Cell signaling, Danvers, MA); SUZ12 and H3K27me3 (Upstate, Billerica, MA); EED and histone H3 lysine 3 trimethylation (H3K4me3) (Abcam, Cambridge, UK); glyceraldehyde-3-phosphate dehydrogenase, cyclin B1, and cadherin E (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Enhancer of zeste homolog 2 overexpression has a role in the development of anaplastic thyroid carcinomas.

doi: 10.1210/jc.2010-1784

Figure Lengend Snippet: FIG. 4. EZH2 suppression inhibits ATC cell growth. A, Growth curve of the stably transfected mass populations of 8505C and FRO phU6 and - phU6-shRNAs. B, Colony-forming assay transfecting ATC cell lines with phU6, phU6-shRNA-962, or -shRNA-2142. C, FACS analysis of 8505C- phU6 or 8505C-shRNA-962 mass populations synchronized by using a double-thymidine block.

Article Snippet: The antibodies used were as follows: EZH2 (Cell signaling, Danvers, MA); SUZ12 and H3K27me3 (Upstate, Billerica, MA); EED and histone H3 lysine 3 trimethylation (H3K4me3) (Abcam, Cambridge, UK); glyceraldehyde-3-phosphate dehydrogenase, cyclin B1, and cadherin E (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Stable Transfection, Transfection, shRNA, Blocking Assay

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Enhancer of zeste homolog 2 overexpression has a role in the development of anaplastic thyroid carcinomas.

doi: 10.1210/jc.2010-1784

Figure Lengend Snippet: FIG. 5. EZH2 depletion suppresses the malignant phenotype of ATC cell lines. A, Soft agar assay to determine anchorage-independent growth of 8505C-phU6 or 8505C-shRNA-962 mass populations. B, Cell migration and invasion assays of 8505C-phU6 and 8505C-shRNA- 962 mass populations.

Article Snippet: The antibodies used were as follows: EZH2 (Cell signaling, Danvers, MA); SUZ12 and H3K27me3 (Upstate, Billerica, MA); EED and histone H3 lysine 3 trimethylation (H3K4me3) (Abcam, Cambridge, UK); glyceraldehyde-3-phosphate dehydrogenase, cyclin B1, and cadherin E (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Soft Agar Assay, shRNA, Migration

Journal: The Journal of clinical endocrinology and metabolism

Article Title: Enhancer of zeste homolog 2 overexpression has a role in the development of anaplastic thyroid carcinomas.

doi: 10.1210/jc.2010-1784

Figure Lengend Snippet: FIG. 6. EZH2 regulates paired-box gene 8 (PAX8) expression. A, qRT-PCR of PAX8 gene expression in human anaplastic thyroid carcinoma tissues (top panel) and in human thyroid carcinoma cell lines (bottom panel). The fold change indicates the relative change in expression levels between ATC and normal thyroid tissues, assuming that the mean value of three normal thyroid samples is equal to 1 (top panel). The fold change indicates the relative change in expression levels between carcinoma cell lines and normal primary thyroid cultures, assuming that the value of the normal thyroid cells is equal to 1 (bottom panel). The scale bars represent the mean SE (n 3). Results are reported as mean expression values of three independent experiments, with error bars indicating SE. B, qRT-PCR and Western blot analyses to quantify restoration of PAX8 mRNA and protein in 8505C and FRO stably transfected with -phU6 and -phU6-shRNAs. Quantitative (C) and semiquantitative (D) PCR analyses of the ChIP from 8505C-phU6 and 8505C-shRNA-962 cells using anti-EZH2 (top panel) and anti-H3K27me3 (K27me3) and anti-H3K4me3 (K4me3) (bottom panel) antibodies. Quantitative and semiquantitative PCR assays were performed amplifying the associated DNA using primers specific for the predicted regulatory region of the PAX8 gene spanning from 2308 to 2371 bp (qPCR) and 2235 to 2622 bp PCR upstream the TSS. IgG indicates the negative control of immunoprecipitation.

Article Snippet: The antibodies used were as follows: EZH2 (Cell signaling, Danvers, MA); SUZ12 and H3K27me3 (Upstate, Billerica, MA); EED and histone H3 lysine 3 trimethylation (H3K4me3) (Abcam, Cambridge, UK); glyceraldehyde-3-phosphate dehydrogenase, cyclin B1, and cadherin E (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Expressing, Quantitative RT-PCR, Gene Expression, Western Blot, Stable Transfection, Transfection, shRNA, Negative Control, Immunoprecipitation

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: 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: 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