Journal: Diagnostic Pathology

Article Title: Enhancer of zeste homologue 2 (EZH2) is a reliable immunohistochemical marker to differentiate malignant and benign hepatic tumors

doi: 10.1186/1746-1596-7-86

Figure Lengend Snippet: EZH2 staining

Article Snippet: The primary antibody was a mouse monoclonal anti-EZH2 (clone 11/EZH2) from BD Biosciences (San Jose CA, USA) (dilution 1:100).

Techniques: Expressing, Wilms Tumor Assay

Journal: Diagnostic Pathology

Article Title: Enhancer of zeste homologue 2 (EZH2) is a reliable immunohistochemical marker to differentiate malignant and benign hepatic tumors

doi: 10.1186/1746-1596-7-86

Figure Lengend Snippet: EZH2 staining in primary liver tumors. A/HCC, nuclear staining in tumor cells, the surrounding liver is negative; B/hepatocellular adenoma, there is no staining; C/CCC, note the unstained nuclei of the non tumorous bile duct in the center¸D/positively stained highly differentiated CCC (Klatskin tumour). Scale bar for the Figure 1:50 μm.

Article Snippet: The primary antibody was a mouse monoclonal anti-EZH2 (clone 11/EZH2) from BD Biosciences (San Jose CA, USA) (dilution 1:100).

Techniques: Staining

Journal: Cancer research

Article Title: Tamoxifen Resistance in Breast Cancer Is Regulated by the EZH2–ERα–GREB1 Transcriptional Axis

doi: 10.1158/0008-5472.CAN-17-1327

Figure Lengend Snippet: EZH2 confers tamoxifen resistance in breast cancer cells. A and B, Comparison of EZH2 mRNA level in breast tumors according to their sensitivities to tamoxifen (A) and Kaplan–Meier analysis (B) of disease-free survival based on EZH2 mRNA levels using GSE9195 cohort (29). Tam-R, tamoxifen-resistant; Tam-S, tamoxifen-sensitive. Red curve, top 50% with high EZH2 level; blue curve, bottom 50% with low EZH2 level. N in brackets, number of patients in each specific group. P value was calculated by Mann–Whitney test (A) and log-rank test (B). C and D, Responses to tamoxifen in originally sensitive MCF-7 cells upon no transfection (MCF-7), stably expressing either empty vector (MCF-7-EV) or HA-tagged EZH2 (MCF-7-EZH2; C) and in TamR MCF-7 cells with EZH2 depletion using two independent shRNAs (shEZH2-1 and -4; D). Cell numbers were counted after 7 days of incubation with indicated concentrations of 4-OHT. Left, Western blot analysis of total cell lysates with indicated antibodies. E, Inhibitory effect of EZH2 inhibitors (GSK343 and EPZ-6438) on proliferation of TamR MCF-7 cells. Cells were maintained in 100 nmol/L 4-OHT and treated with DMSO (Veh.), 5 μmol/L GSK343 or EPZ-6438, then collected at indicated time points for cell counting. Left, Western blot analysis of total cell lysates and nuclear extract. Data were presented as mean ± SEM of triplicates. *, P < 0.05; **, P < 0.001, and P values in C–E were calculated by two-tailed t test.

Article Snippet: IHC analysis and quantification Paraffin-embedded breast cancer samples from patients and xenograft tumors were subjected to IHC staining with primary antibodies as follows: EZH2 (dilution 1:200; #NCL-L-EZH2; Novocastra), GREB1 (dilution 1:400; #MABS62; EMD Millipore), ERα (dilution 1:75; #NCL-L-ER-6F11; Novocastra), Ki67 (dilution 1:400; #9027; Cell Signaling Technology), and cleaved caspase-3 (dilution 1:100, #9661S; Cell Signaling Technology).

Techniques: MANN-WHITNEY, Transfection, Stable Transfection, Expressing, Plasmid Preparation, Incubation, Western Blot, Cell Counting, Two Tailed Test

Journal: Cancer research

Article Title: Tamoxifen Resistance in Breast Cancer Is Regulated by the EZH2–ERα–GREB1 Transcriptional Axis

doi: 10.1158/0008-5472.CAN-17-1327

Figure Lengend Snippet: EZH2 regulates an ERα-associated transcriptional profile in favor of endocrine resistance. A, GSEA analysis of differentially expressed genes upon EZH2 knockdown in TamR MCF-7 cells. P values were determined by Kolmogorov–Smirnov test. B, Unsupervised hierarchical cluster analysis of genes that were changed when MCF-7 was treated with E2 (E2 vs. Veh.) or E2 plus 4-OHT (E2 + 4-OHT vs. E2) using GSE25316 dataset (19) and genes that are differentially expressed upon EZH2 knockdown in TamR MCF-7 cells (shEZH2 vs. shCtrl). Color scale bar indicates the log2 of differential gene expression from the lowest (blue) to the highest (red) level. C, Scatter plot showing transcriptional changes of tamoxifen-regulated genes upon the antiestrogen treatment (fold change ≥ 1.5, Padj < 0.05) in either control cells (x-axis) or EZH2-overexpressing MCF-7 cells (y-axis). D, Functional annotations of EZH2-regulated genes in TamR MCF-7 cells. Annotations that are associated with endocrine therapy resistance were highlighted in red. Blue bar, numbers of genes overlapped within each indicated gene set; red line, adjusted P value of each functional category. E and F, Quantitative RT-PCR showing expression changes of ERα target genes in MCF-7 cells upon EZH2 overexpression (E) and TamR MCF-7 cells upon EZH2 depletion (F) with the treatment of either ethanol (Veh.) or 100 nmol/L 4-OHT for 6 hours. G and H, ChIP-qPCR results at cis-regulatory elements near XBP1 gene in EZH2-overexpressing MCF-7 cells (G) and EZH2-silenced TamR MCF-7 cells (H) with the treatment of 1 μmol/L 4-OHT for 45 minutes. Data were plotted as means ± SEM of replicates after being normalized to GAPDH (mRNA) or KIAA0066 (ChIP-qPCR) as the internal control. *, P < 0.05 and **, P < 0.001 by two-sided t test.

Article Snippet: IHC analysis and quantification Paraffin-embedded breast cancer samples from patients and xenograft tumors were subjected to IHC staining with primary antibodies as follows: EZH2 (dilution 1:200; #NCL-L-EZH2; Novocastra), GREB1 (dilution 1:400; #MABS62; EMD Millipore), ERα (dilution 1:75; #NCL-L-ER-6F11; Novocastra), Ki67 (dilution 1:400; #9027; Cell Signaling Technology), and cleaved caspase-3 (dilution 1:100, #9661S; Cell Signaling Technology).

Techniques: Expressing, Functional Assay, Quantitative RT-PCR, Over Expression

Journal: Cancer research

Article Title: Tamoxifen Resistance in Breast Cancer Is Regulated by the EZH2–ERα–GREB1 Transcriptional Axis

doi: 10.1158/0008-5472.CAN-17-1327

Figure Lengend Snippet: EZH2 epigenetically silences GREB1 expression via DNA methylation. A, ROC curve analysis indicating the correlation of EZH2-regulated genes that are involved in ERα signaling with tamoxifen responses in GSE9195 and GSE12093 cohorts (29, 30). Blue dots, EZH2-repressed genes; red dots, EZH2-activated genes. B and C, Upregulation of GREB1 at both mRNA (B) and protein (C) levels upon shEZH2 in TamR MCF-7 cells. D, Clinical relevance of DNA methylation at a specified area within GREB1 promoter. Top, duplicates of DNA methylation profiling in MCF-7 cells and three distinct endocrine-resistant, MCF-7–derived cell lines (26). TamR, tamoxifen-resistant; MCF7X, estrogen deprivation resistant; FASR, fulvestrant resistant. Location of the CpG sites displaying differential patterns is highlighted in gray. Middle, heat map depicting the correlation of DNA methylation signals at each specified probe for every single patient with GREB1 expression in TCGA data (28). Bottom, correlation between the methylation levels of the underlined probes with pathologic stages concerning the involvement of regional lymph node. P value was calculated by Fisher exact test. E, Decrease in DNA methylation intensity at GREB1 promoter region upon EZH2 depletion in TamR MCF-7 cells. Pyrosequencing was performed at locus highlighted in D. F, Restoration of EZH2 overexpression-induced repression of GREB1 expression by 5-Aza. Control (EV) or EZH2-overexpressing (EZH2) MCF-7 cells were treated with DMSO (Veh.) or increasing concentrations of 5-Aza (1, 5, and 10 μmol/L) for 72 hours. G, RT-qPCR showing transcript levels of GREB1 and ESR1 upon EZH2 knockdown with or without 5-Aza treatment. Stable TamR MCF-7 clones were treated with DMSO (Veh.) or 5-Aza (5 μmol/L) for 120 hours. Data are presented as means ± SEM of replicates. *, P < 0.05 and **, P < 0.001 by two-sided t test.

Article Snippet: IHC analysis and quantification Paraffin-embedded breast cancer samples from patients and xenograft tumors were subjected to IHC staining with primary antibodies as follows: EZH2 (dilution 1:200; #NCL-L-EZH2; Novocastra), GREB1 (dilution 1:400; #MABS62; EMD Millipore), ERα (dilution 1:75; #NCL-L-ER-6F11; Novocastra), Ki67 (dilution 1:400; #9027; Cell Signaling Technology), and cleaved caspase-3 (dilution 1:100, #9661S; Cell Signaling Technology).

Techniques: Expressing, DNA Methylation Assay, Derivative Assay, Methylation, Over Expression, Quantitative RT-PCR, Clone Assay

Journal: Cancer research

Article Title: Tamoxifen Resistance in Breast Cancer Is Regulated by the EZH2–ERα–GREB1 Transcriptional Axis

doi: 10.1158/0008-5472.CAN-17-1327

Figure Lengend Snippet: GREB1 blocks the activation of tamoxifen-liganded ERα. A and B, Alleviation of the inhibitory effects of tamoxifen on ERα activity upon GREB1 knockdown. Upon transient transfection with control siRNA (siCtrl) or siRNA pool targeting GREB1 (siGREB1), MCF-7 cells were hormone deprived for 3 days and then treated with ethanol (Veh.), 10 nmol/L E2, or 10 nmol/L E2 plus 100 nmol/L 4-OHT for 6 hours. Total cell lysates were prepared for immunoblotting (A) or total RNA was subjected to qRT-PCR (B). mRNA levels were normalized to GAPDH and are presented as mean ± SEM of replicates. C, Binding of ERα coregulators near target genes in GREB1-depleted breast cancer cells. GREB1 was silenced in MCF-7 cells by two independent shRNAs (shGREB1-1 and shGREB1-2), and the stable clones were treated with ethanol (Veh.), 100 nmol/L E2, or 1 μmol/L 4-OHT for 45 minutes after hormone deprivation for 3 days. Quantitative PCR was performed and are presented as mean ± SEM of replicates. D, Essential role of GREB1 in mediating the effects of EZH2 on conferring resistance to tamoxifen. Stable clones of EZH2-depleted (shEZH2) TamR MCF-7 cells were infected with two independent shGREB1 and treated with 4-OHT at indicated concentrations for 7 days before cell numbers were counted. Bottom, Western blot analysis confirming the knockdown efficiencies of shEZH2 and shGREB1. Statistical difference was calculated in comparison with the data from EZH2-depleted cells. E, ChIP-qPCR showing reverse of EZH2 depletion-induced chromatin redistribution of ERα coregulators upon GREB1 knockdown. Data are presented as mean ± SEM of triplicates. *, P < 0.05 and **, P < 0.001 by two-tailed t test.

Article Snippet: IHC analysis and quantification Paraffin-embedded breast cancer samples from patients and xenograft tumors were subjected to IHC staining with primary antibodies as follows: EZH2 (dilution 1:200; #NCL-L-EZH2; Novocastra), GREB1 (dilution 1:400; #MABS62; EMD Millipore), ERα (dilution 1:75; #NCL-L-ER-6F11; Novocastra), Ki67 (dilution 1:400; #9027; Cell Signaling Technology), and cleaved caspase-3 (dilution 1:100, #9661S; Cell Signaling Technology).

Techniques: Activation Assay, Activity Assay, Transfection, Western Blot, Quantitative RT-PCR, Binding Assay, Clone Assay, Real-time Polymerase Chain Reaction, Infection, Two Tailed Test

Journal: Cancer research

Article Title: Tamoxifen Resistance in Breast Cancer Is Regulated by the EZH2–ERα–GREB1 Transcriptional Axis

doi: 10.1158/0008-5472.CAN-17-1327

Figure Lengend Snippet: Pharmacologic inhibition of EZH2 represents a promising therapeutic strategy for TamR breast cancer. A, Efficacy of EZH2 inhibitors on the growth of TamR xenograft tumors at the indicated dose for a course of 35 days. Data are presented as mean tumor volume ± SEM. B, Effects of EZH2 inhibitors on body weights of mice receiving the treatment. Data are presented as mean ± SEM. C, Characterization of TamR MCF-7 xenograft tumors with histologic analysis by hematoxylin and eosin (H&E) staining and IHC staining of Ki67 and cleaved caspase-3. Scale bars, 100 μm. D, Western blot analysis of indicated proteins in TamR xenograft tumors. Bottom, quantitative densitometry of protein levels of GREB1, EZH2, and ERα. Number 1–12, numbers of randomly selected xenograft tumors with four individual samples per group. Data are presented as mean ± SEM. *, P < 0.05 and **, P < 0.01 by two-sided t test.

Article Snippet: IHC analysis and quantification Paraffin-embedded breast cancer samples from patients and xenograft tumors were subjected to IHC staining with primary antibodies as follows: EZH2 (dilution 1:200; #NCL-L-EZH2; Novocastra), GREB1 (dilution 1:400; #MABS62; EMD Millipore), ERα (dilution 1:75; #NCL-L-ER-6F11; Novocastra), Ki67 (dilution 1:400; #9027; Cell Signaling Technology), and cleaved caspase-3 (dilution 1:100, #9661S; Cell Signaling Technology).

Techniques: Inhibition, Staining, Immunohistochemistry, Western Blot

Journal: Cancer research

Article Title: Tamoxifen Resistance in Breast Cancer Is Regulated by the EZH2–ERα–GREB1 Transcriptional Axis

doi: 10.1158/0008-5472.CAN-17-1327

Figure Lengend Snippet: Negative correlation between EZH2 and GREB1 in ER+ breast cancer samples receiving tamoxifen as adjuvant treatment. A, Correlation between EZH2 and GREB1 transcript levels in patient samples. The black curve shows the correlation coefficients and the red shows the P value. The dotted red line represents P = 0.05. B, IHC staining showing nuclear staining of EZH2, GREB1, and ERα levels in representative ER+ breast tumors. Scale bar, 50 μm. C, Correlation between EZH2 and GREB1 protein levels based on their IHC scores. P values were calculated using χ2 analysis. D, Percentages of each GREB1 IHC score within the specific groups of samples that are stratified on the basis of either EZH2 (left) or ERα (right) IHC scores. Med, medium. P values were calculated using Fisher exact analysis. E, Quantification of EZH2, GREB1, and ERα IHC staining in tamoxifen-sensitive and TamR breast cancer patients. N in brackets, number of patients in each specific group. P values were determined by two-tailed Mann–Whitney test.

Article Snippet: IHC analysis and quantification Paraffin-embedded breast cancer samples from patients and xenograft tumors were subjected to IHC staining with primary antibodies as follows: EZH2 (dilution 1:200; #NCL-L-EZH2; Novocastra), GREB1 (dilution 1:400; #MABS62; EMD Millipore), ERα (dilution 1:75; #NCL-L-ER-6F11; Novocastra), Ki67 (dilution 1:400; #9027; Cell Signaling Technology), and cleaved caspase-3 (dilution 1:100, #9661S; Cell Signaling Technology).

Techniques: Immunohistochemistry, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Cancer research

Article Title: Tamoxifen Resistance in Breast Cancer Is Regulated by the EZH2–ERα–GREB1 Transcriptional Axis

doi: 10.1158/0008-5472.CAN-17-1327

Figure Lengend Snippet: EZH2-mediated regulation of ERα signaling is highly clinical relevant to tamoxifen response. A and B, Kaplan–Meier curve showing disease-free survival rates in ER+ breast tumors according to both EZH2 and GREB1 levels using GSE9195 and GSE12093 datasets (refs. 29, 30; A) and in 130 ER+ breast cancer samples for IHC staining (B). +/−, expression level of top 50% (+) or bottom 50% (−) of the gene. C and D, Expression levels of gene signature that is repressed by EZH2 and involved in ERα signaling in tamoxifen-sensitive (Tam-S) or tamoxifen-resistant (Tam-R) breast tumors (C) and their prognostic power (D) using GSE9195 cohort (29). Figure legends represent the same meaning as in Fig. 1A and B. E, Model depicting the development tamoxifen resistance driven by the EZH2–ERα–GREB1 transcriptional axis. T, tamoxifen; Me, methylation.

Article Snippet: IHC analysis and quantification Paraffin-embedded breast cancer samples from patients and xenograft tumors were subjected to IHC staining with primary antibodies as follows: EZH2 (dilution 1:200; #NCL-L-EZH2; Novocastra), GREB1 (dilution 1:400; #MABS62; EMD Millipore), ERα (dilution 1:75; #NCL-L-ER-6F11; Novocastra), Ki67 (dilution 1:400; #9027; Cell Signaling Technology), and cleaved caspase-3 (dilution 1:100, #9661S; Cell Signaling Technology).

Techniques: Immunohistochemistry, Expressing, Methylation

Journal: Oncology Letters

Article Title: Efficacy of extracts of Celastrus orbiculatus in suppressing migration and invasion by inhibiting the EZH2/ROCK1 signaling pathway in human nasopharyngeal carcinoma

doi: 10.3892/ol.2018.8149

Figure Lengend Snippet: Primers for reverse transcription-quantitative polymerase chain reaction analysis.

Article Snippet: Primary antibodies used were: Rabbit anti-EZH2 (cat. no. ab186006; 1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-ROCK1 (cat. no. ab45171; 1:2,000; Abcam) and rabbit anti-β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Polymerase Chain Reaction, Sequencing

Journal: Oncology Letters

Article Title: Efficacy of extracts of Celastrus orbiculatus in suppressing migration and invasion by inhibiting the EZH2/ROCK1 signaling pathway in human nasopharyngeal carcinoma

doi: 10.3892/ol.2018.8149

Figure Lengend Snippet: Protein expression levels of EZH2 and ROCK1 from 5–8F cells determined using western blot analysis. 5–8F cells were treated with COE (0, 12.5, 25 and 50 µg/ml) or 2 µM DZNeP. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05. EZH2, enhancer of zeste homolog 2; ROCK1, Rho-associated coiled coil-containing protein kinase 1; COE, Celastrus orbiculatus extract; DZNeP, 3-Deazaneplanocin A.

Article Snippet: Primary antibodies used were: Rabbit anti-EZH2 (cat. no. ab186006; 1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-ROCK1 (cat. no. ab45171; 1:2,000; Abcam) and rabbit anti-β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Oncology Letters

Article Title: Efficacy of extracts of Celastrus orbiculatus in suppressing migration and invasion by inhibiting the EZH2/ROCK1 signaling pathway in human nasopharyngeal carcinoma

doi: 10.3892/ol.2018.8149

Figure Lengend Snippet: Reverse transcription-quantitative polymerase chain reaction determination of relative EZH2 and ROCK1 mRNA expression in 5–8F cells. Results are presented as the mean ± standard deviation of three independent experiments. *P<0.05. (A) Relative EZH2 mRNA expression. (B) Relative ROCK1 mRNA expression. EZH2, enhancer of zeste homolog 2; ROCK1, Rho-associated coiled coil-containing protein kinase 1; DZNeP, 3-Deazaneplanocin A; COE, Celastrus orbiculatus extract.

Article Snippet: Primary antibodies used were: Rabbit anti-EZH2 (cat. no. ab186006; 1:1,000; Abcam, Cambridge, MA, USA), rabbit anti-ROCK1 (cat. no. ab45171; 1:2,000; Abcam) and rabbit anti-β-actin (cat. no. 4970; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Standard Deviation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Recurrence-Associated Long Non-coding RNA LNAPPCC Facilitates Colon Cancer Progression via Forming a Positive Feedback Loop with PCDH7

doi: 10.1016/j.omtn.2020.03.017

Figure Lengend Snippet: LNAPPCC Activates PCDH7 Expression via Binding EZH2 (A) RNA pull-down assay using biotin-labeled LNAPPCC was undertaken to detect the interaction between LNAPPCC and EZH2. (B) RIP assay using EZH2 specific antibody was undertaken to detect the binding between EZH2 and LNAPPCC. (C) Schematic outlining the predicted G-rich motif in LNAPPCC and the wild-type (WT) and mutated LNAPPCC sequences used for RIP with anti-EZH2. LNAPPCC-Mut contains a deletion of the G-rich motif. (D) After transient transfection of LNAPPCC-WT or LNAPPCC-Mut overexpression plasmids into HCT116 cells, RIP assay was undertaken using EZH2 specific antibody. (E) ChIP assay using EZH2 and H3K27me3 specific antibodies was undertaken in LNAPPCC stably overexpressed and control SW620 cells to detect the effects of LNAPPCC overexpression on the binding of EZH2 to PCDH7 promoter and H3K27me3 level at PCDH7 promoter. (F) ChIP assay using EZH2 and H3K27me3 specific antibodies was undertaken in LNAPPCC stably silenced and control HCT116 cells to detect the effects of LNAPPCC silencing on the binding of EZH2 to PCDH7 promoter and H3K27me3 level at PCDH7 promoter. (G) PCDH7 mRNA and protein levels in LNAPPCC stably overexpressed and control HCT116 cells were detected by quantitative real-time PCR and western blot. (H) PCDH7 mRNA and protein levels in LNAPPCC stably overexpressed and control SW620 cells were detected by quantitative real-time PCR and western blot. (I) PCDH7 mRNA and protein levels in LNAPPCC stably silenced and control HCT116 cells were detected by quantitative real-time PCR and western blot. Data are presented as mean ± SD based on at least three independent biological repeats. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ns, not significant, by Student’s t test (B, D, E, G, and H) or one-way ANOVA followed by Dunnett’s multiple comparisons test (F and I).

Article Snippet: After being transferred into polyvinylidene fluoride (PVDF) membrane (Millipore), the blots were incubated with primary antibodies against EZH2 (1:500; 07-689; Millipore), PCDH7 (1:200; ab139274; Abcam), phospho-ERK1/2 (1:1,000; #4370; Cell Signaling Technology), ERK1/2 (1:1,000; #4695; Cell Signaling Technology), phospho-c-FOS (1:1,000; #5348; Cell Signaling Technology), c-FOS (1:1,000; #2250; Cell Signaling Technology), or GAPDH (1:10,000; T0004; Affinity).

Techniques: Expressing, Binding Assay, Pull Down Assay, Labeling, Transfection, Over Expression, Stable Transfection, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Annals of Translational Medicine

Article Title: H3K27me3 induces multidrug resistance in small cell lung cancer by affecting HOXA1 DNA methylation via regulation of the lncRNA HOTAIR

doi: 10.21037/atm.2018.10.21

Figure Lengend Snippet: H3K27me3 was related to resistance to anticancer drug treatment. (A) H3K27me3 and EZH2 levels in SCLC tissue and normal lung tissue were detected by IHC staining; (B) H3K27me3 and EZH2 levels in multidrug-resistant cells and sensitive cells were detected by Western blotting; (C) H3K27me3 and EZH2 levels after treatment with different concentrations of EI1 and GSK J4; (D) IC50 values were measured with CCK-8 assays after EI1-treated and GSK J4-treated cells were exposed to CDDP, ADM, and VP-16; (E) cell apoptosis was determined by flow cytometric analysis in EI1-treated and GSK J4-treated cells after drug exposure. The results are presented as the mean ± SD. *P<0.05, **P<0.01, compared with the control group.

Article Snippet: Sample sections were incubated with primary antibodies directed against EZH2 (Cell Signaling Technology, #5426, USA) and H3K27me3 (Cell Signaling Technology, #9733, USA) at 4 °C overnight.

Techniques: Immunohistochemistry, Western Blot, CCK-8 Assay, Control

Journal: Annals of Translational Medicine

Article Title: H3K27me3 induces multidrug resistance in small cell lung cancer by affecting HOXA1 DNA methylation via regulation of the lncRNA HOTAIR

doi: 10.21037/atm.2018.10.21

Figure Lengend Snippet: Protein levels of EZH2 and increased H3K27me3 were revealed by Western blotting in SCLC cells and after treatment with E1I and GSK J4. Band intensities were measured by densitometry; EZH2 levels were normalized to β-actin expression, and H3K27me3 levels were normalized to H3 expression.

Article Snippet: Sample sections were incubated with primary antibodies directed against EZH2 (Cell Signaling Technology, #5426, USA) and H3K27me3 (Cell Signaling Technology, #9733, USA) at 4 °C overnight.

Techniques: Western Blot, Expressing

Journal: Annals of Translational Medicine

Article Title: H3K27me3 induces multidrug resistance in small cell lung cancer by affecting HOXA1 DNA methylation via regulation of the lncRNA HOTAIR

doi: 10.21037/atm.2018.10.21

Figure Lengend Snippet: EZH2 and H3K27me3 levels were upregulated by HOTAIR. (A,C) HOTAIR overexpression and knockdown were validated by RT-PCR; (B,D) H3K27me3 and EZH2 enrichment were measured with Western blotting assays of the HOTAIR-overexpressing and HOTAIR-knockdown cells. The results are presented as the mean ± SD. **P<0.01, compared with the control group.

Article Snippet: Sample sections were incubated with primary antibodies directed against EZH2 (Cell Signaling Technology, #5426, USA) and H3K27me3 (Cell Signaling Technology, #9733, USA) at 4 °C overnight.

Techniques: Over Expression, Knockdown, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control

Journal: Annals of Translational Medicine

Article Title: H3K27me3 induces multidrug resistance in small cell lung cancer by affecting HOXA1 DNA methylation via regulation of the lncRNA HOTAIR

doi: 10.21037/atm.2018.10.21

Figure Lengend Snippet: Schematic illustration of HOTAIR/EZH2-mediated H3K27me3 and HOXA1 DNA methylation in chemoresistant SCLC cells.

Article Snippet: Sample sections were incubated with primary antibodies directed against EZH2 (Cell Signaling Technology, #5426, USA) and H3K27me3 (Cell Signaling Technology, #9733, USA) at 4 °C overnight.

Techniques: DNA Methylation Assay