expression plasmid  (Sino Biological)

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

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: Afterward, 25 ng of cDNA was mixed with 0.65 µL of predesigned Taqman TM gene expression assays 6-FAM-3ʹBHQ-1 EZH2 (Applied Biosystems, Darmstadt, Germany), 0.65 µL of in-house 5-VIC-3ʹBHQ-1 GAPDH for normalization, 6.5 µL of master mix, and 3.2 µL of water.

Techniques: Expressing, Derivative Assay, Amplification, 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: Afterward, 25 ng of cDNA was mixed with 0.65 µL of predesigned Taqman TM gene expression assays 6-FAM-3ʹBHQ-1 EZH2 (Applied Biosystems, Darmstadt, Germany), 0.65 µL of in-house 5-VIC-3ʹBHQ-1 GAPDH for normalization, 6.5 µL of master mix, and 3.2 µL of water.

Techniques: Immunofluorescence, Staining, Control

Journal: Cell Reports Medicine

Article Title: Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor

doi: 10.1016/j.xcrm.2023.101354

Figure Lengend Snippet: Anti-transcription activity of γPNA-NLS in lymphoma cells (A–C) Relative fold change in c-Myc and EZH2 after treatment with PBS and the indicated γPNAs (8 μM) for 24 h. (D–F) Percentage of viability of lymphoma cells treated with PBS, γPNA1-NLS, γPNA2-NLS, and Scr-γPNA3-NLS for 24 h. (G and H) Fold change in live and early apoptotic lymphoma cells after 24 h treatment with γPNA2-NLS and Scr-γPNA3-NLS (8 μM). (I) Relative change in c-My c in U2932 cells treated with the indicated γPNA2-NLS doses in comparison with Scr-γPNA3-NLS. (J and K) Representative WBs of c-MYC protein in lymphoma cells (top) after 24 h treatment with PBS (control), γPNA2-NLS, and Scr-γPNA3-NLS (8 μM). Graphs represent the quantification of c-MYC protein fold change relative to PBS. (A–K) Graphs show mean ± SEM (n = 3); p value for unpaired two-sample t test. (L) WB representing levels of γH2AX protein in γPNA2-NLS and Scr-γPNA3-NLS (8 μM) after 24 h relative to PBS. (M) Representative immunofluorescence images of HeLa cells stained with γH2AX antibody after 24 h treatment with γPNA2-NLS (8 μM) and bleomycin. Scale bar, 50 μm.

Article Snippet: TaqManTM Gene Expression Assay (FAM) Inventoried human EZH2 (Hs00544830) , Thermofisher Scientific , Cat# 4331182.

Techniques: Activity Assay, Comparison, Control, Immunofluorescence, Staining

Journal: Cell Reports Medicine

Article Title: Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor

doi: 10.1016/j.xcrm.2023.101354

Figure Lengend Snippet: Efficacy of anti-transcription γPNA-NLS in lymphoma xenograft mice (A) IVIS images of U2932 xenografts post γPNA5-NLS-T administration at the indicated time points (5 mg/kg dose, i.v.). (B) IVIS images of tumor and organs, including kidneys (K), lungs (Lu), liver (L), spleen (S), intestine (In), and bone marrow (BM), after γPNA5-NLS-T administration. (C) Confocal images of γPNA5-NLS-T treated xenografts. Blue, DAPI. (D and E) c-MYC and EZH2 protein levels (D) and quantification (E) in control and γPNA2-NLS-treated (25 mg/kg, i.v.) U2932 xenografts after 24 h (control, n = 6; γPNA2-NLS, n = 7). (F and G) c-MYC and EZH2 protein levels (F) and quantification (G) in control and γPNA2-NLS-treated (5 mg/kg, i.t.) Raji xenografts after 24 h (control, n = 5; γPNA2-NLS, n = 6). (E and G) Graphs show mean ± SEM; p value for unpaired two-sample t test. (H) Tumor growth curve of U2932 xenograft mice treated with γPNA2-NLS and Scr-γPNA3-NLS (5 mg/kg) (saline, n = 10; γPNA2-NLS, n = 9; Scr-γPNA3-NLS, n = 8). Mean ± SEM; p value for two-way ANOVA. (I) Immunohistochemistry of tumors, including Ki67 and caspase-3, staining in U2932 xenografts post survival study. Scale bar, 50 μm. (J) Serum chemistry analysis of U2932 xenografts post survival. Graphs show mean ± SEM (n = 3).

Article Snippet: TaqManTM Gene Expression Assay (FAM) Inventoried human EZH2 (Hs00544830) , Thermofisher Scientific , Cat# 4331182.

Techniques: Control, Saline, Immunohistochemistry, Staining

Journal: Cell Reports Medicine

Article Title: Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor

doi: 10.1016/j.xcrm.2023.101354

Figure Lengend Snippet: Efficacy of γPNA2-NLS in combination with HDACis and chemotherapeutic drugs (A) Percentage viability of U2932 cells treated with the indicated γPNA-NLS, HDAC inhibitors, and combination at 1, 2, and 4 days. (B) Fold change of c-Myc and EZH2 gene expression in treated U2932 cells on day 4. (C) WB analysis representing c-MYC and EZH2 protein levels in U2932 cells on day 4. (D) Fold change in c-Myc and EZH2 levels in Raji cells after 24 h. (E) Percentage of viability of U2932 cells treated with doses of CHOP or in combination with γPNA2-NLS after 24 h. (A, B, D, and E) Graphs show mean ± SEM (n = 3); p value for unpaired two-sample t test. (F) Workflow representing the in vivo study plan to evaluate the efficacy of γPNA2-NLS with romidepsin (top) and tumor growth curve of U2932 xenografts after treatment with romidepsin and combination of romidepsin with γPNA2-NLS (bottom). Vehicle, n = 5; romidepsin, n = 8; γPNA2-NLS+romidepsin, n = 8. (G) Representative immunohistochemistry images of H&E, Ki67, and caspase-3 staining in U2932 xenografts post survival. (F and G) Graphs show mean ± SEM (n = 3); p value for two-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.0001. (H) Workflow representing the in vivo study plan to evaluate γPNA2-NLS efficacy with CHOP (top) and tumor growth curve of U2932 xenografts treated with CHOP and γPNA2-NLS+CHOP (bottom). Control, n = 6; CHOP, n = 7; γPNA2-NLS+CHOP, n = 6. (I) Representative immunohistochemistry images of H&E, Ki67, and caspase-3 staining in control, CHOP-, and γPNA2-NLS+CHOP-treated U2932 tumors post survival. (G and I) Scale bars, 100 μm (H&E) and 50 μm (immunohistochemistry).

Article Snippet: TaqManTM Gene Expression Assay (FAM) Inventoried human EZH2 (Hs00544830) , Thermofisher Scientific , Cat# 4331182.

Techniques: Gene Expression, In Vivo, Immunohistochemistry, Staining, Control

Journal: Cell Reports Medicine

Article Title: Antitumor efficacy of a sequence-specific DNA-targeted γPNA-based c-Myc inhibitor

doi: 10.1016/j.xcrm.2023.101354

Figure Lengend Snippet:

Article Snippet: TaqManTM Gene Expression Assay (FAM) Inventoried human EZH2 (Hs00544830) , Thermofisher Scientific , Cat# 4331182.

Techniques: Plasmid Preparation, Recombinant, Binding Assay, Staining, Red Blood Cell Lysis, DC Protein Assay, Gene Expression, DNA Purification, Purification, Reverse Transcription, Luminex, RNA Sequencing, Amplification, Oligonucleotide Synthesis, Software, Fluorescence, Microscopy, Real-time Polymerase Chain Reaction, Electroporation, Multiplex Assay