ha ezh2  (Addgene inc)

Journal: Science advances

Article Title: BRD9 functions as a methylarginine reader to regulate AKT-EZH2 signaling.

doi: 10.1126/sciadv.ads6385

Figure Lengend Snippet: Fig. 5. BRD9 regulates H3K27me3 levels through the AKT1-EZH2 axis. (A) IB analysis of WCL derived from MDA-MB-231 cells infected with lentivirus of sgGFP or sg- BRD9. (B) IB analysis of WCL derived from MDA-MB-231 cells treated with indicated doses of dBRD9 for 16 hours. (C) IB analysis of WCL derived from MDA-MB-231 cells infected with lentivirus of sgGFP or sgBRD9. Cells were treated with 5 μM GSK126 for 16 hours before harvesting. (D) IB analysis of WCL derived from MDA-MB-231 cells treated with indicated doses of I-BRD9 for 16 hours. (E) IB analysis of WCL derived from MDA-MB-231 cells stably expressing EV or Myr-AKT1 and depleted of BRD9 in- fected with lentivirus sgGFP (−) or sgBRD9 (+). (F) IB analysis of WCL derived from control (sgGFP) or AKT1-depleted MDA-MB-231 cells. Cells were treated with 5 μM I- BRD9 for 16 hours before harvesting. (G) RT-qPCR analysis of mRNA levels of select genes in MDA-MB-231 cells treated with DMSO or 5 μM GSK126 for 48 hours. Data are shown as means ± SD of n = 3 biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.001, Student’s t test.

Article Snippet: Myr- AKT1 (64606) and HA- EZH2 (173717) were purchased from Addgene.

Techniques: Derivative Assay, Infection, Stable Transfection, Expressing, Control, Quantitative RT-PCR

Journal: Science advances

Article Title: BRD9 functions as a methylarginine reader to regulate AKT-EZH2 signaling.

doi: 10.1126/sciadv.ads6385

Figure Lengend Snippet: Fig. 6. Combined treatment of BRD9 and EZH2 inhibitors leads to synergistic growth inhibition of breast cancer. (A) Inhibition of cell viability and dose-response matrixes analyzed by SynergyFinder. MDA-MB-231 cells were treated with the indicated doses of I-BRD9 and GSK126 for 96 hours prior to analysis of cell viability. (B) MDA- MB-231 cells treated with I-BRD9 or GSK126 were subjected to cell proliferation assays. Data are shown as means ± SD of n = 3 biological replicates. ***P < 0.001, two-way ANOVA and Tukey post hoc test. (C) MDA-MB-231 cells treated with I-BRD9 or GSK126 were subjected to colony formation assays. Representative images are shown. (D) Quantification of colonies in (C). Data are shown as means ± SD of n = 3 biological replicates. *P < 0.05 and ***P < 0.001, one-way ANOVA and Tukey post hoc test. (E) Schematic of a mouse xenograft assay to evaluate the antitumor effects of I-BRD9 and GSK126. (F) Tumor growth curve upon treatment of I-BRD9 and GSK126. Data are shown as means ± SEM of n = 6 mice for each group. *P < 0.05, two-way ANOVA and Tukey post hoc test. (G and H) Dissected tumors were weighed. Data are shown as the means ± SEM of n = 6 tumors for each group. *P < 0.05, one-way ANOVA and Tukey post hoc test. (I) Representative images of TUNEL assays in xenograft tumors in (G). (J) Schematic depicting the function of the BRD9-AKT-EZH2 axis in regulating transcription and tumor growth.

Article Snippet: Myr- AKT1 (64606) and HA- EZH2 (173717) were purchased from Addgene.

Techniques: Inhibition, Xenograft Assay, TUNEL Assay

Journal: Cancers

Article Title: Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

doi: 10.3390/cancers16213660

Figure Lengend Snippet: Copy number variation of EZH2 gene in melanoma. ( A ) EZH2 gain status in different cancer types from the cBioPortal database. ( B ) Copy number variation of EZH2 gene in 547 melanoma samples. *** p < 0.001. ( C ) Distribution pie chart of EZH2 copy number ( n = 547). According to the EZH2 copy number, it is divided into four subgroups. Subgroup 1: copy number ≤ 2; subgroup 2: 2 < copy number ≤ 3; subgroup 3: 3 < copy number ≤ 4; subgroup 4: copy number > 4. ( D ) Correlation of EZH2 gain status with its mRNA expression in melanoma samples from the cBioPortal database ( n = 367). * p < 0.05, *** p < 0.001. ( E ) Association of EZH2 copy number gain with expression levels in melanoma subtypes ( n = 183). The left panel represents the percentage of cases with or without EZH2 copy number gain across different expression levels (0, 1, 2, 3). The right panel illustrates the proportion of expression levels in different melanoma subtypes: acral melanoma (AM), mucosal melanoma (MM), and cutaneous melanoma (CM). The staining score for each sample, counting the intensity of the staining, was graded as 0, 1, 2, and 3 (“0” as negative, and “3” as the strongest).

Article Snippet: pCMV3-HA vector containing the human EZH2 coding sequence (HA- EZH2 ) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Expressing, Staining

Journal: Cancers

Article Title: Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

doi: 10.3390/cancers16213660

Figure Lengend Snippet: EZH2 amplification in melanoma.

Article Snippet: pCMV3-HA vector containing the human EZH2 coding sequence (HA- EZH2 ) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Amplification

Journal: Cancers

Article Title: Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

doi: 10.3390/cancers16213660

Figure Lengend Snippet: Correlation of EZH2 gain to clinicopathologic features of mucosal melanoma.

Article Snippet: pCMV3-HA vector containing the human EZH2 coding sequence (HA- EZH2 ) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques:

Journal: Cancers

Article Title: Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

doi: 10.3390/cancers16213660

Figure Lengend Snippet: Overall survival of melanoma patients in relation to EZH2 copy number variations. Comparison of the overall survival (OS) of tumors with different EZH2 copy number levels in melanoma subtypes was conducted by the Kaplan–Meier method. ( A ) all melanoma cases, n = 547. ( B ) acral melanoma cases, n = 252. ( C ) cutaneous melanoma cases, n = 147. ( D ) mucosal melanoma cases, n = 148. EZH2 -No gain was considered as samples with copy numbers less than or equal to 2.0. EZH2 gain was considered as samples with copy numbers greater than 2.0.

Article Snippet: pCMV3-HA vector containing the human EZH2 coding sequence (HA- EZH2 ) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Comparison

Journal: Cancers

Article Title: Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

doi: 10.3390/cancers16213660

Figure Lengend Snippet: Univariate and multivariate analysis of EZH2 gain and clinicopathologic factors associated with overall survival in mucosal melanoma.

Article Snippet: pCMV3-HA vector containing the human EZH2 coding sequence (HA- EZH2 ) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Mutagenesis

Journal: Cancers

Article Title: Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

doi: 10.3390/cancers16213660

Figure Lengend Snippet: Loss of EZH2 inhibits MM cell proliferation and progression in vitro and in vivo. ( A ) The expression of EZH2 was detected by RT-qPCR (top row) or Western blot assay (bottom row) after the knockdown of EZH2 . The data are presented as the mean ± SEM. n = 3, *** p < 0.001. ( B ) HMV-II, LM-MEL-53, and GAK cells with stable depletion of EZH2 or control were grown for 5 days, with cell numbers counted every day by CCK-8 assays. The changes in cell numbers were compared to day 0, and the mean ± SEM from 3 experiments was plotted. *** p < 0.001. ( C ) The proliferative abilities of stably EZH2 -depleted HMVII cells were measured with Ki-67 staining assay. Three experiments were conducted with mean ± SEM of percentage of Ki-67-positive cells plotted. Scale bar: 100 μm. *** p < 0.001. ( D ) The average sizes of xenograft tumors were measured every 3 days and plotted ( n = 5, error bars indicate mean ± SEM). * p < 0.05, *** p < 0.001.

Article Snippet: pCMV3-HA vector containing the human EZH2 coding sequence (HA- EZH2 ) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: In Vitro, In Vivo, Expressing, Quantitative RT-PCR, Western Blot, Knockdown, Control, CCK-8 Assay, Stable Transfection, Staining

Journal: Cancers

Article Title: Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

doi: 10.3390/cancers16213660

Figure Lengend Snippet: EZH2 knockdown enhances ferroptotic sensitivity in MM cells. ( A ) Cell viability response to treatment with apoptosis and ferroptosis inducers in cells with EZH2 knockdown. Cell viability was assessed after treatment with a range of concentrations of apoptosis inducer staurosporine (left panel), apoptosis inducer actinomycin D (middle panel), and ferroptosis inducer erastin (right panel). The mean ± SEM from 3 experiments was plotted. ( B ) Representative images of EZH2 knockdown effects on the viability of 3D spheroids formed by LM-MEL-53 cells in response to 4 µM erastin, as indicated by GFP fluorescence. Scale bars: 200 µm. ( C ) Bar graph showing viability of LM-MEL-53 cells with EZH2 knockdown, treated with 4 µM erastin or 4 µM erastin and 4 µM Ferrostatin-1. The data are presented as the mean ± SEM. n = 3, * p < 0.05, *** p < 0.001. ( D ) Bar graph demonstrating intracellular glutathione levels in EZH2 -depleted HMV-II and LM-MEL-53 cells. The data are presented as the mean ± SEM. n = 3, ** p < 0.01. ( E ) Lipid peroxidation was measured by flow cytometry after 5 μM CellROX Deep Red staining in EZH2 -depleted cells. The data are presented as the mean ± SEM. n = 3, *** p < 0.001. ( F ) The level of malondialdehyde in cells was determined by using a malondialdehyde kit after the knockdown of EZH2 . The data are presented as the mean ± SEM. n = 3, *** p < 0.001. ( G ) TEM was used to detect the mitochondrial morphology of ferroptotic cells. Scale bars: 1 μm (left column), and 500 nm (right column).

Article Snippet: pCMV3-HA vector containing the human EZH2 coding sequence (HA- EZH2 ) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Knockdown, Fluorescence, Flow Cytometry, Staining

Journal: Cancers

Article Title: Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

doi: 10.3390/cancers16213660

Figure Lengend Snippet: Depletion of EZH2 stimulates ferroptosis through decreased SLC7A11 . ( A ) Volcano plot displaying differential expression from RNA-seq data. Red points indicate upregulated genes, blue points show downregulated genes, and grey points represent genes without significant changes. Vertical dashed lines mark fold change thresholds, and the horizontal line indicates the p -value cutoff for significance. ( B ) Venn diagram showing the significant overlap between RNA-seq data and FerrDb database. SCORE values were derived from FerrD. The SCORE values were listed below the Venn diagram, with SLC7A11 as the top hit ( C , D ). The expression of SLC7A11 was detected by RT-qPCR ( C ) or Western blot assay ( D ) with EZH2 knockdown. The data are presented as the mean ± SEM. n = 3, * p < 0.05, ** p < 0.01, *** p < 0.001. ( E ) Representative phase-contrast images of EZH2 -depleted LM-MEL-53 cells, with or without SLC7A11 re-expression, treated with 4 μM erastin or 4 µM erastin and 4 µM Ferrostatin-1 ( n = 3). Scale bar: 200 μm.

Article Snippet: pCMV3-HA vector containing the human EZH2 coding sequence (HA- EZH2 ) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Expressing, RNA Sequencing Assay, Derivative Assay, Quantitative RT-PCR, Western Blot, Knockdown

Journal: Cancers

Article Title: Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

doi: 10.3390/cancers16213660

Figure Lengend Snippet: EZH2 -mediated SLC7A11 upregulation is regulated by KLF14 . ( A ) Venn diagram of RNA-seq and ChIP-seq related genes showing that 73 genes were found to be potential target genes of EZH2 . ( B ) Heatmap showing the 6 transcription factors of the 73 potential target genes of EZH2 . ( C ) The binding of EZH2 to KLF14 promoter was detected after EZH2 overexpression by ChIP-qPCR. IgG as a negative control. The data are presented as the mean ± SEM. n = 3, ns, not significant, *** p < 0.001. ( D ) The protein level of KLF14 was detected with EZH2 depletion. ( E ) The protein level of SLC7A11 after transfection of KLF14 siRNA. ( F ) Schematic representation of the predicted KLF14 binding site within the SLC7A11 promoter. ( G ) The binding of KLF14 to SLC7A11 promoter was detected after KLF14 overexpression by ChIP-qPCR. IgG as a negative control. The data are presented as the mean ± SEM. n = 3, ns, not significant, *** p < 0.001. ( H ) Luciferase assay measuring SLC7A11 promoter activity before and after KLF14 binding site deletion in the absence or presence of KLF14 . Luciferase activities were normalized to Renilla luciferase activity. The data are presented as the mean ± SEM. n = 3, *** p < 0.001. ( I ) Western blot analysis of EZH2, KLF14, and SLC7A11 protein expression following dual knockdown of EZH2 and KLF14 . ( J ) The correlations between EZH2 and SLC7A11 protein expression in MM patients were analyzed by Pearson correlation analysis ( n = 55). Representative images from immunohistochemical staining of EZH2 and SLC7A11 protein expression. The staining score for each sample, counting the intensity of the staining, was graded as 0, 1, 2, and 3 (“0” as negative, and “3” as the strongest). Scale bar: 100 μm.

Article Snippet: pCMV3-HA vector containing the human EZH2 coding sequence (HA- EZH2 ) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Binding Assay, Over Expression, Negative Control, Transfection, Luciferase, Activity Assay, Western Blot, Expressing, Knockdown, Immunohistochemical staining, Staining

Journal: Cancers

Article Title: Enhancer of Zeste Homolog 2 Protects Mucosal Melanoma from Ferroptosis via the KLF14-SLC7A11 Signaling Pathway

doi: 10.3390/cancers16213660

Figure Lengend Snippet: Effects of combined EZH2 inhibitor and ferroptosis inducer treatment in MM. ( A – C ) Evaluation of combinatorial treatment effects on melanoma organoid models. ( A ) Schematic representation of the experimental design for combinatorial treatment with MS8815 and erastin on melanoma organoid models. ( B ) Representative images of melanoma organoids treated with DMSO (vehicle control), MS8815, erastin, and a combination of MS8815 and erastin, showing morphological changes ( n = 3). Scale bar: 100 µm. ( C ) Quantification of cellular ATP levels as a measure of cell viability post-treatment (*** p < 0.001 compared to vehicle control). ( D – F ) Effects of combined MS8815 and erastin on tumor growth in a MM PDX model. ( D ) Tumor volume was measured over an 18-day period. ( E ) Tumors were weighed and plotted ( n = 5). Data were expressed as mean ± SEM. * p < 0.05, *** p < 0.001. ( F ) Representative images from H&E and immunohistochemical staining of Ki-67 protein expression. Scale bars: 100 μm.

Article Snippet: pCMV3-HA vector containing the human EZH2 coding sequence (HA- EZH2 ) was purchased from Sino Biological (HG11337-CY; Sino Biological, Beijing, China).

Techniques: Control, Immunohistochemical staining, Staining, Expressing