Journal: Cancer Biology & Therapy

Article Title: USP44 promotes chemotherapeutic drug resistance of triple negative breast cancer through EZH2 protein stability

doi: 10.1080/15384047.2025.2529652

Figure Lengend Snippet: USP44 produces chemotherapy resistance to TNBC via EZH2. (a) BT-549 cells with or without OE-USP44 were lysed using RIPA and the lysates were pretreated with protein A/G beads at 4 C for one hour. The cell lysates were then treated with protein A/G beads containing anti-USP44 (5 µg) antibodies at 4 C overnight. Then, the supernatant was discarded and loading buffer was added to crack the magnetic beads. 10% SDS-PAGE gel was used to load 100 μg of protein and stained with Coomassie brilliant blue. Mass spectrometry showed the spectrogram of EZH2 in BT-549 cells pulled down by the USP44 antibody. (b) Expression of EZH2 in TNBC cells with or without OE-USP44/shUSP44 was detected by western blot. C) TNBC cells with OE-USP44 were treated with different concentration of DOX with or without GSK126 (2 μM) for 48 hours. Then, cell viability was measured with the CCK-8 kit ( n = 3). (d) Under the same conditions as (c), Cleaved PARP and H3K27ME3 protein was detected by western blot in two TNBC cell lines. β-actin was used as the loading control for western blot. Boxplots are shown as mean ± SD. * p < .05; ** p < .01; *** p < .001.

Article Snippet: The antibodies used in this study include mouse anti-human USP44 (Santa cruz, sc -377,203), rabbit anti-human EZH2 (Proteintech 21,800–1-AP) for co-immunoprecipitation and mouse anti-human EZH2 (Abcam, ab283270), rabbit anti-human PARP1(Proteintech 13,371–1-AP), mouse anti human UB (CST, 3936), anti-FLAG tag (sigma, F1084) and rabbit anti-human β- Actin (Abclonal, AC026).

Techniques: Magnetic Beads, SDS Page, Staining, Mass Spectrometry, Expressing, Western Blot, Concentration Assay, CCK-8 Assay, Control

Journal: Cancer Biology & Therapy

Article Title: USP44 promotes chemotherapeutic drug resistance of triple negative breast cancer through EZH2 protein stability

doi: 10.1080/15384047.2025.2529652

Figure Lengend Snippet: USP44 stabilizes EZH2 through deubiquitinase activity. (a) Extracts from MDA-MB-231 cells were isolated for co-immunoprecipitation using an anti-USP44 antibody or anti-EZH2 antibody. Specifically, MDA-MB-231 cells were lysed using RIPA and the lysates were pretreated with protein A/G beads at 4 C for one hour. The cell lysates were then treated with protein A/G beads containing anti-USP44 (5 µg) or anti-EZH2 (5 µg) antibodies at 4°C overnight. Then, the supernatant was discarded and loading buffer was added to crack the magnetic beads. The interaction of endogenous USP44 and EZH2 was tested. Normal mouse IgG was used as a control. (b) BT-549 cells with or without OE-USP44 were treated with CHX (25 µg/mL) and harvested at the indicated times (0,2,4,8 hours), then protein levels of USP44 amd EZH2 were analyzed by western blot. (c) Similarly, the same BT-549 cells with or without shUSP44 were treated as above, then protein levels of USP44 amd EZH2 were analyzed by western blot. (d) BT-549 cells with or without shUSP44 were treated with or without MG132 (1 μM) for 24 hours. The protein expression levels of USP44 and EZH2 were confirmed followed by western blot. β-actin was used as the loading control. (e) pLent-puro-ubiquitin plasmids was transfected into BT-549 cells with or without OE-USP44. After continuing to incubate for 48 h, the cells were lyzed with RIPA lysate and MG132 (10 μM) was added 2 hours before this step. Then the lysates were pretreated with protein A/G beads at 4 C for one hour. The cell lysates were then treated with protein A/G beads containing anti-EZH2 (5 µg) antibody at 4°C overnight. Then, the supernatant was discarded and loading buffer was added to crack the magnetic beads. USP44 ubiquitination was detected by western blot with anti-UB antibody. The protein expression levels of USP44 and EZH2 in BT-549 cells were confirmed. Boxplots are shown as mean ± SD. * p < .05; ** p < .01; *** p < .001.

Article Snippet: The antibodies used in this study include mouse anti-human USP44 (Santa cruz, sc -377,203), rabbit anti-human EZH2 (Proteintech 21,800–1-AP) for co-immunoprecipitation and mouse anti-human EZH2 (Abcam, ab283270), rabbit anti-human PARP1(Proteintech 13,371–1-AP), mouse anti human UB (CST, 3936), anti-FLAG tag (sigma, F1084) and rabbit anti-human β- Actin (Abclonal, AC026).

Techniques: Activity Assay, Isolation, Immunoprecipitation, Magnetic Beads, Control, Western Blot, Expressing, Ubiquitin Proteomics, Transfection

Journal: Cancer Biology & Therapy

Article Title: USP44 promotes chemotherapeutic drug resistance of triple negative breast cancer through EZH2 protein stability

doi: 10.1080/15384047.2025.2529652

Figure Lengend Snippet: In vivo validity of targeting EZH2 to sensitize TNBC cells to DOX. (a) Nude BALB/C mice were subcutaneously xenografted with 4T1cells (1×10 cells) into the flanks and injected intraperitoneally with DOX (3 mg/kg) and GSK126 (100 mg/kg) alone or in combination every two days for consecutive 14 days. (b) After 14 days, the mice were executed and the xenograft tumors were isolated. (c) Tumor weighing analysis. (d) Tumor growth curves for each group. (e) Representative IHC images showing H3K27me3 and Ki-67 expression in tumors from each group of mice. Boxplots are shown as mean ± SD. * p < .05; ** p < .01; *** p < .001.

Article Snippet: The antibodies used in this study include mouse anti-human USP44 (Santa cruz, sc -377,203), rabbit anti-human EZH2 (Proteintech 21,800–1-AP) for co-immunoprecipitation and mouse anti-human EZH2 (Abcam, ab283270), rabbit anti-human PARP1(Proteintech 13,371–1-AP), mouse anti human UB (CST, 3936), anti-FLAG tag (sigma, F1084) and rabbit anti-human β- Actin (Abclonal, AC026).

Techniques: In Vivo, Injection, Isolation, Expressing

Journal: Cancer Biology & Therapy

Article Title: USP44 promotes chemotherapeutic drug resistance of triple negative breast cancer through EZH2 protein stability

doi: 10.1080/15384047.2025.2529652

Figure Lengend Snippet: A graphic abstract of USP44-EZH2 axis regulating DOX resistance in TNBC.

Article Snippet: The antibodies used in this study include mouse anti-human USP44 (Santa cruz, sc -377,203), rabbit anti-human EZH2 (Proteintech 21,800–1-AP) for co-immunoprecipitation and mouse anti-human EZH2 (Abcam, ab283270), rabbit anti-human PARP1(Proteintech 13,371–1-AP), mouse anti human UB (CST, 3936), anti-FLAG tag (sigma, F1084) and rabbit anti-human β- Actin (Abclonal, AC026).

Techniques:

Journal: Journal of experimental & clinical cancer research : CR

Article Title: EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A.

doi: 10.1186/s13046-023-02855-2

Figure Lengend Snippet: Fig. 1 Coordinated expression of EZH2 and TOP2A in HCC. (A) Heatmap of genes correlated with EZH2 in cancers analyzed by the Oncomine database. (B) Volcano plot showing the differentially expressed genes of EZH2 in HCC by reanalyzing the RNA-seq data in the TCGA dataset using the limma pack age in R software. (C and D) Correlation analysis of EZH2 and TOP2A in HCC (C) and 31 tumors (including ACC, BLCA, BRCA, CESC, CHOL, COAD, DLBC, ESCA, GBM, HNSC, KICH, KIRC, KIRP, LAML, LGG, LIHC, LUAD, LUSC, MESO, OV, PAAD, PCPG, PRAD, READ, SARC, SKCM, STAD, TGCT, THCA, THYM, UCEC, UCS, and UVM) in TCGA (D). (E) TOP2A expression in normal tissues (n = 50), HCC tissues with low EZH2 expression (n = 185), and HCC tissues with high EZH2 expression (n = 186) by reanalyzing the RNA-seq data of HCC in the TCGA dataset using R software v4.0.3. (F) Western blot and (G) RT-qPCR analysis of EZH2 and TOP2A expression patterns in HCC cell lines. (H) Protein levels of EZH2 and TOP2A in HCC tissues and paired paracancerous tissues (n = 47) analyzed by IHC. GSEA of RNA-seq data from TCGA of EZH2 high expression versus EZH2 low expression (J) and TOP2A high expression versus TOP2A low expression using the Reactome cellular senescence gene set annotated in R-HSA-2,559,583. NES, normalized enrichment score. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The slides were stained with anti-human EZH2 (Cell Signaling Technology, 5246 S, 1:50) and TOP2A (Cell Signaling Technology, 12,286 S, 1:50) antibodies.

Techniques: Expressing, RNA Sequencing, Software, Western Blot, Quantitative RT-PCR

Journal: Journal of experimental & clinical cancer research : CR

Article Title: EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A.

doi: 10.1186/s13046-023-02855-2

Figure Lengend Snippet: Fig. 2 Inhibition of EZH2 impairs growth and induces senescence of HCC cells both in vitro and in vivo. (A) Representative images of colony formation and SA-β-gal staining of cells. (B) mRNA levels of EZH2 and senescence markers p15, p16, and p21 in BEL7404 and SMMC7721 cells. (C) Representative images of tumors in nude mice subcutaneously inoculated with BEL7404 cells stably expressing pLKO.1-shEZH2s and pLKO.1-control (seven mice in each group). (D and E) The tumor volume (D) and weight (E) in nude mice subcutaneously inoculated with BEL7404 cells stably expressing pLKO.1-shEZH2s and pLKO.1-control. (F) Representative images of H&E, Ki67, TOP2A, and SA-β-gal staining of tumor tissues. (G) The cell rate of Ki67-positive cells in tumors. (H) The cell rate of TOP2A-positive cells in tumors. (I) The cell rate of SA-β-gal-positive cells in tumors. (J) mRNA levels of EZH2, TOP2A, and senescence markers p15, p16, and p21 in tumors in nude mice subcutaneously inoculated with BEL7404 cells. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The slides were stained with anti-human EZH2 (Cell Signaling Technology, 5246 S, 1:50) and TOP2A (Cell Signaling Technology, 12,286 S, 1:50) antibodies.

Techniques: Inhibition, In Vitro, In Vivo, Staining, Stable Transfection, Expressing, Control

Journal: Journal of experimental & clinical cancer research : CR

Article Title: EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A.

doi: 10.1186/s13046-023-02855-2

Figure Lengend Snippet: Fig. 5 EZH2 acts as a positive regulator of TOP2A by promoting H3K27me3-mediated epigenetic silencing of miR-139-5p. (A) Protein levels of TOP2A and H3K27me3 in cells treated with the EZH2 inhibitors UNC1999 and EPZ005687. (B and C) Protein levels (B) and mRNA levels (C) of EZH2 and TOP2A in cells transfected with EZH2-targeted siRNAs. (D and E) mRNA levels of miR-139-5p in cells treated with EZH2 inhibitors UNC1999 and EPZ005687 (D) and in cells transfected with EZH2 targeted siRNAs (E). (F) mRNA levels of miR-139-5p in cells transfected with EED- and SUZ12- targeted siRNAs. (G) ChIP‒qPCR analysis of the enrichment of EZH2 and H3K27me3 on the promoter region of miR-139-5p in HCC cell lines. IgG was used as a negative control and H3 was used as a positive control. (H) Western blot analysis of TOP2A and H3K27me3 in HCC cells transfected with inhibitor NC and miR139-5p inhibitor and treated with DMSO, UNC1999, or EPZ005687. (I) Western blot analysis of TOP2A, EZH2 and H3K27me3 in HCC cells transfected with inhibitor NC and miR139-5p inhibitor and transfected with NC or siEZH2. (J) Western blot analysis of TOP2A, Dicer, EZH2 and H3K27me3 in HCC cells transfected with NC, siEZH2, siDicer, and siEZH2 and siDicer. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The slides were stained with anti-human EZH2 (Cell Signaling Technology, 5246 S, 1:50) and TOP2A (Cell Signaling Technology, 12,286 S, 1:50) antibodies.

Techniques: Transfection, Negative Control, Positive Control, Western Blot

Journal: Journal of experimental & clinical cancer research : CR

Article Title: EZH2-H3K27me3-mediated silencing of mir-139-5p inhibits cellular senescence in hepatocellular carcinoma by activating TOP2A.

doi: 10.1186/s13046-023-02855-2

Figure Lengend Snippet: Fig. 7 The expression profiles and correlation with prognosis of the EZH2/miR-139-5p/TOP2A axis in HCC. (A) The heatmap shows the expression profiles of EZH2 and TOP2A across tumor samples and paired normal tissues in 31 tumors in the TCGA database. E-N for EZH2 expression in normal tissues, E-T for EZH2 expression in tumor tissues, T-N for TOP2A expression in normal tissues, T-T for TOP2A expression in tumor tissues. (B) The expression of EZH2 and TOP2A in HCC cohorts (GSE14520 and GSE6764). (C) Protein levels of EZH2 and TOP2A in ten pairs of HCC tissues and para-carcinoma tissues. (D) mRNA levels of EZH2, TOP2A and miR-139-5p in ten pairs of HCC tissues and para-carcinoma tissues. (E) The expression of miR-139-5p in HCC in the TCGA database. (F) The expression correlation of EZH2, TOP2A and miR-139-5p in ten pairs of HCC tissues and para-carcinoma tissues. (G) Overall survival rate of HCC from the TCGA database analyzed according to the mRNA levels of EZH2 and TOP2A. HCC patients were divided into high and low groups according to the median mRNA expression level of EZH2 and TOP2A, respectively. HH for EZH2-High/TOP2A-High (n = 158); EZH2-High/TOP2A-Low (n = 27); EZH2- Low/TOP2A-High (n = 27); LL for EZH2-Low/TOP2A-Low (n = 158). (H) The correlation of EZH2, TOP2A, and miR-139-5p with overall survival (OS), relapse- free survival (RFS), progression-free survival (PFS), and disease-free survival (DFS) in patients with HCC. *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: The slides were stained with anti-human EZH2 (Cell Signaling Technology, 5246 S, 1:50) and TOP2A (Cell Signaling Technology, 12,286 S, 1:50) antibodies.

Techniques: Expressing

Journal: Journal of Clinical and Translational Hepatology

Article Title: MALAT1-mediated EZH2 Recruitment to the GFER Promoter Region Curbs Normal Hepatocyte Proliferation in Acute Liver Injury

doi: 10.14218/JCTH.2021.00391

Figure Lengend Snippet: The H3K27me3 methylation peak in the GFER promoter region was predicted by the UCSU database (A); RIP assays was performed to identify the interaction between MALAT1 and EZH2 (B), *** p <0.001 vs. the IgG group; ChIP-PCR was used to detect the enrichment of EZH2 and H3H27me3 expression in the GFER promoter region (C), * p <0.05, ** p <0.01 vs. the oe-NC or sh-NC group; After transfection of oe-EZH2 or sh-EZH2 into HL7702 cells, western blotting quantified EZH2, H3K27me3 and GFER expression (D), * p <0.05, ** p <0.01 vs. the oe-NC or sh-NC group. n =3. ChIP, chromatin immunoprecipitation; NC, negative control; RIP, RNA immunoprecipitation.

Article Snippet: Briefly, HL7702 cells were lysed in 100 µL lysis buffer containing protease and RNase inhibitors, and then the protein lysate was incubated with rabbit anti-human EZH2 antibody (ab186006 1:500; Abcam) at 4°C for 30 m or anti-IgG antibody (ab109489, 1:100; Abcam) as the control.

Techniques: Methylation, Expressing, Transfection, Western Blot, Chromatin Immunoprecipitation, Negative Control, Immunoprecipitation

Journal: Journal of Clinical and Translational Hepatology

Article Title: MALAT1-mediated EZH2 Recruitment to the GFER Promoter Region Curbs Normal Hepatocyte Proliferation in Acute Liver Injury

doi: 10.14218/JCTH.2021.00391

Figure Lengend Snippet: After introduction of sh-MALAT1, oe-MALAT1, sh-EZH2, oe-EZH2, sh-GFER, and oe-GFER for 24 h, HL7702 cells were subjected to LPS treatment for 16 h. Phosphorylated AMPK and mTOR were assayed by western blotting (A–C), * p <0.05, ** p <0.01 vs. the LPS + oe-NC or LPS + sh-NC group. HL7702 cells were transfected with oe-MALAT1 or oe-NC for 24 h and then stimulated with the AMPK inhibitor Compound C for 1 h, and treated with LPS. Phosphorylated AMPK and mTOR levels were determined by Western blotting (D, F); ALT (G), AST (H) and LDH (I); Cell proliferation was determined by CCK-8 assays (J) and EdU staining (K); Cell apoptosis was assayed by TUNEL staining (L), Determination of MDA (M), GSH (N), and SOD (O) in cells. * p <0.05 vs. LPS + oe-NC or LPS + oe-MALAT1 group. n =3. AST, aspartic transaminase; ALT, alanine aminotransferase; GSH, glutathione; LDH, lactic dehydrogenase; LPS, lipopolysaccharide; MDA, malondialdehyde; NC, negative control; SOD, superoxide dismutase.

Article Snippet: Briefly, HL7702 cells were lysed in 100 µL lysis buffer containing protease and RNase inhibitors, and then the protein lysate was incubated with rabbit anti-human EZH2 antibody (ab186006 1:500; Abcam) at 4°C for 30 m or anti-IgG antibody (ab109489, 1:100; Abcam) as the control.

Techniques: Western Blot, Transfection, CCK-8 Assay, Staining, TUNEL Assay, Negative Control

Journal: PLoS ONE

Article Title: Promotion of Glioblastoma Cell Motility by Enhancer of Zeste Homolog 2 (EZH2) Is Mediated by AXL Receptor Kinase

doi: 10.1371/journal.pone.0047663

Figure Lengend Snippet: A EZH2 expression (red) in human glioma sections of different WHO grades: astrocytoma grade II (top), astrocytoma grade III (middle), glioblastoma (bottom). Magnification 200x, inset 400x. B Box-plot of EZH2 expression in human brain tumors of increasing malignancy (WHO grade II: n = 10, mean = 6, SD = 18.97; WHO grade III: n = 15, mean = 40.67, SD = 56.91; GBM: n = 12, mean = 135, SD = 98.59; r 2 = 0.385, P = 0.024). C EZH2 expression (red) in glioblastoma sections in close proximity to necrotic areas. Arrows indicate the necrotic area. Magnification 100x. D EZH2 mRNA expression in A172, LN18, U87MG human malignant glioma cells, S24, T269 human glioma-initiating cells (GIC), human astrocytes and human mesenchymal stem cells (MSC) measured by qRT-PCR. E EZH2 protein expression in A172, LN18, U87MG human malignant glioma cells, S24, T269 human GIC, human astrocytes and human MSC. Tubulin served as loading control. Asterisk indicates * (p<0.05). Error bars indicate s.e.m.

Article Snippet: Pre-treatment was followed by incubation with either rabbit anti-human EZH2 antibody (1∶100; Invitrogen) or rabbit anti-human AXL (1∶50; Sigma-Aldrich) at 37°C for 32 min. Incubation was followed by Ventana standard signal amplification, UltraWash, counterstaining with one drop of hematoxylin for 4 min and one drop of bluing reagent for 4 min. For visualization, ultraView™Universal DAB Detection Kit (Ventana Medical Systems) was used.

Techniques: Expressing, Quantitative RT-PCR

Journal: PLoS ONE

Article Title: Promotion of Glioblastoma Cell Motility by Enhancer of Zeste Homolog 2 (EZH2) Is Mediated by AXL Receptor Kinase

doi: 10.1371/journal.pone.0047663

Figure Lengend Snippet: A EZH2 transcript expression was decreased 24 h after si EZH2 treatment (left). Western blot showing EZH2 protein expression, 120 h after knockdown by siRNA (right). Tubulin served as loading control. B Cell cycle analysis of U87MG glioma cells 120 h after specific knockdown of EZH2 (si EZH2 , right) or scrambled control (siC, left). C Invasion of U87MG glioma cells with transient EZH2 knockdown (lower panel, black bar) through a matrigel-coated boyden chamber in comparison to control (upper panel, white bar). D Representative dot plots and corresponding analysis of Annexin-V-FITC/DAPI co-staining of U87MG glioma cells untreated or treated for 120 h with 10 µM DZNep. The lower left quadrants represent the living cells (low Annexin-V-FITC-/DAPI-signal), the lower right quadrants represents early apoptosis (low DAPI- and strong Annexin-V-FITC-signal) and the upper right late apoptotic/necrotic cells (double-stained cells). E H3K27me3 methylation was strongly decreased in whole cell lysates of U87MG glioma cells after treatment with 5 µM DZNep or after specific knockdown of EZH2 for 120 h. Tubulin served as loading control. F Cell cycle analysis of U87MG glioma cells untreated or treated for 120 h with 500 nM and 5 µM DZNep. G Analysis of nestin expression in S24 glioma-initiating cells untreated (left) or treated with 5 µM DZNep for 120 h (right) by flow cytometry. H Matrigel boyden chamber assay of U87MG glioma cells untreated (upper panel, white bar) and treated (lower panel, black bar) with 5 µM DZNep. Asterisk indicates * (p<0.05). Error bars indicate s.e.m.

Article Snippet: Pre-treatment was followed by incubation with either rabbit anti-human EZH2 antibody (1∶100; Invitrogen) or rabbit anti-human AXL (1∶50; Sigma-Aldrich) at 37°C for 32 min. Incubation was followed by Ventana standard signal amplification, UltraWash, counterstaining with one drop of hematoxylin for 4 min and one drop of bluing reagent for 4 min. For visualization, ultraView™Universal DAB Detection Kit (Ventana Medical Systems) was used.

Techniques: Expressing, Western Blot, Cell Cycle Assay, Staining, Methylation, Flow Cytometry, Boyden Chamber Assay

Journal: PLoS ONE

Article Title: Promotion of Glioblastoma Cell Motility by Enhancer of Zeste Homolog 2 (EZH2) Is Mediated by AXL Receptor Kinase

doi: 10.1371/journal.pone.0047663

Figure Lengend Snippet: A mRNA expression of various known EZH2 target genes 120 h after siRNA mediated EZH2 knockdown in U87MG glioma cells. B Table of known EZH2 target genes, which were regulated as indicated in response to EZH2 knockdown compared to control in U87MG glioma cells. C Heatmap of the ten most regulated genes in response to specific knockdown of EZH2 by siRNA after 120 h in U87MG cells. Upregulated genes are indicated in blue, downregulated genes in yellow. D AXL protein expression in the human glioma cell line U87MG (left) and the human GIC S24 (right) by flow cytometry. The cells were stained with an AXL specific antibody: blue or an isotype control: red. E AXL mRNA expression levels in U87MG human glioma cells incubated for 120 h with scrambled control siRNA (siC) or EZH2 specific siRNA (si EZH2 ). F Analysis of AXL protein expression in U87MG glioma cells treated with siC (solid line) or si EZH2 (dashed line) for 120 h stained with a AXL specific antibody: blue, isotype control: red. Asterisk indicates * (p<0.05). Error bars indicate s.e.m. NS: not significant.

Article Snippet: Pre-treatment was followed by incubation with either rabbit anti-human EZH2 antibody (1∶100; Invitrogen) or rabbit anti-human AXL (1∶50; Sigma-Aldrich) at 37°C for 32 min. Incubation was followed by Ventana standard signal amplification, UltraWash, counterstaining with one drop of hematoxylin for 4 min and one drop of bluing reagent for 4 min. For visualization, ultraView™Universal DAB Detection Kit (Ventana Medical Systems) was used.

Techniques: Expressing, Flow Cytometry, Staining, Incubation

Journal: PLoS ONE

Article Title: Promotion of Glioblastoma Cell Motility by Enhancer of Zeste Homolog 2 (EZH2) Is Mediated by AXL Receptor Kinase

doi: 10.1371/journal.pone.0047663

Figure Lengend Snippet: A AXL mRNA expression of U87MG human glioma cells treated with 5 µM DZNep for 120 h in comparison to control. B Analysis of AXL protein expression in U87MG glioma cells untreated (solid line) or treated with 5 µM DZNep for 120 h (dashed line) stained with an AXL specific antibody (blue) or an isotype control antibody (red). C Comparison of genes with decreased (upper Venn-diagram) or increased (lower Venn diagram) expression after 120 h of EZH2 knockdown (purple) or DZNep treatment (blue) of U87MG glioma cells. Cutoff: 1.5 fold change. D AXL mRNA expression of U87MG human malignant glioma cells after 96 h treatment with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-aza) (black bars) or DMSO (white bar). E EZH2 and AXL mRNA expression of U87MG glioma cells after the stimulation with indicated concentrations of the histone deacetylase inhibitors suberoylanilide hydroxamic acid (SAHA) or trichostatin A (TSA) (black bars) or DMSO (white bars) for 24 h. Asterisk indicates * (p<0.05). Error bars indicate s.e.m.

Article Snippet: Pre-treatment was followed by incubation with either rabbit anti-human EZH2 antibody (1∶100; Invitrogen) or rabbit anti-human AXL (1∶50; Sigma-Aldrich) at 37°C for 32 min. Incubation was followed by Ventana standard signal amplification, UltraWash, counterstaining with one drop of hematoxylin for 4 min and one drop of bluing reagent for 4 min. For visualization, ultraView™Universal DAB Detection Kit (Ventana Medical Systems) was used.

Techniques: Expressing, Staining, DNA Methylation Assay, Histone Deacetylase Assay