anti human ezh2  (Cell Signaling Technology Inc)

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: NPJ Precision Oncology

Article Title: Combined inhibition of EZH2 and CDK4/6 perturbs endoplasmic reticulum-mitochondrial homeostasis and increases antitumor activity against glioblastoma

doi: 10.1038/s41698-024-00653-3

Figure Lengend Snippet: A EZH2 expression levels (qPCR) in primary tumors and patient-derived GBM cell lines (left) (ns not significant), as well as FISH analysis (right) to detect EZH2 in GBM cells. FISH was performed using the ZytoLight FISH Cytology implementation Kit according to the manufacturer’s protocol using the following probes: ZytoLight SPEC CUX1 (green)/EZH2 (red)/CEN 7 (blue) Triple Color Probe. The criteria for gene amplification were defined as the presence of either four (or more) gene signals or more than 2.5 times as many gene signals as centromere signals of the related chromosome. B protein abundance in four patient-derived GBM cell lines. Representative immunofluorescence images are shown. Nuclei were stained with DAPI (blue), actin filaments with Phalloidin green (green), EZH2 using an Alexa Fluor® 647-conjugated secondary antibody (red). Single-channel and merged fluorescence are presented (scale bar: 20 μm). C – E Influence of GSK126 alone and in combination with abemaciclib on the viability of GBM cells. C Concentration-response relationships to GSK126 for one (72 h) and two (2 × 72 h) treatment cycles to determine the IC 20 and IC 50 concentrations. D Presented is the response to GSK126 (10 µM), abemaciclib (1 µM) and the combination ( C , D ) of both after 2 treatment cycles with 72 h each; n = 3, mean ± s.d. One-way ANOVA (Tukey’s multiple comparisons test); *p < 0.05; **p < 0.01; ****p < 0.0001 (comparison between control and test group); # p < 0.05; ## p < 0.001; #### p < 0.0001 (comparison between testing groups). E The Bliss independence model was used to assess additive or synergistic effects in the combination compared to each monotherapy after 2 × 72 h of treatment. Created with Biorender.com.

Article Snippet: Specific antibody (Ab) staining was done at RT using anti-human EZH2 (Cat# 14-9867-82) or anti-human CDK4 (Cat# MA5-41178, both from Thermo Fisher Scientific, stock: 1 mg/mL, 1:50, 2 h), followed by washing and incubation with the secondary antibody (Cat# 405322, anti-rabbit IgG (H + L), F(ab’)2 Fragment Alexa Fluor® 647, Biolegend, San Diego, CA, USA, 1:250, 60 min).

Techniques: Expressing, Derivative Assay, Amplification, Quantitative Proteomics, Immunofluorescence, Staining, Fluorescence, Concentration Assay, Comparison, Control

Journal: NPJ Precision Oncology

Article Title: Combined inhibition of EZH2 and CDK4/6 perturbs endoplasmic reticulum-mitochondrial homeostasis and increases antitumor activity against glioblastoma

doi: 10.1038/s41698-024-00653-3

Figure Lengend Snippet: Immunofluorescence for assessment of A , B EZH2 and C , D CDK4 protein levels in 2D cell cultures with or without treatment (GSK126 10 µM, abemaciclib 1 µM, combination, 2 × 72 h). Nuclei were stained with DAPI (blue), actin filaments with Phalloidin green (green), EZH2/CDK4 using an Alexa Fluor® 647-conjugated secondary antibody (red). B , D The quantification is presented as the x-fold change (integrated density) relative to the control (DMSO), which was set to =1 (dotted line); n = 3, mean ± s.d. Kruskal–Wallis test (Dunn’s multiple comparisons test).

Article Snippet: Specific antibody (Ab) staining was done at RT using anti-human EZH2 (Cat# 14-9867-82) or anti-human CDK4 (Cat# MA5-41178, both from Thermo Fisher Scientific, stock: 1 mg/mL, 1:50, 2 h), followed by washing and incubation with the secondary antibody (Cat# 405322, anti-rabbit IgG (H + L), F(ab’)2 Fragment Alexa Fluor® 647, Biolegend, San Diego, CA, USA, 1:250, 60 min).

Techniques: Immunofluorescence, Staining, 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. 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