ezh2 primary antibody  (Vector Laboratories)

Journal: JCI Insight

Article Title: Inhibition of histone methyltransferase EZH2 for immune interception of colorectal cancer in Lynch syndrome

doi: 10.1172/jci.insight.177545

Figure Lengend Snippet: ( A ) Western blot analysis was performed in the lysates from colonic mucosal stripping of VCMsh2T Hu mice ( N = 3/group): control and GSK treatment with quantification shown in bar graph below blots. Histone H3 modification was assessed using anti-H3K27me3 ( P = 0.0003), anti-H3K9me3 ( P = 0.0091), anti-H3K36me3 ( P = 0.6534), and anti-H3K4me3 ( P = 0.0005) antibodies with histone H3 as loading control. Stemness and proliferation were measured via anti-LGR5 ( P = 0.3583), anti-EPCAM ( P = 0.0001), anti-GATA3 ( P = 0.2559), and Ki67 antibodies ( P = 0.0572). Anti-EZH2 ( P = 0.3091) was probed to assess efficacy of GSK503 activity in the colonic mucosa. The loading control for each nonhistone blot was β-actin. Quantification was performed using ImageJ, and density was normalized to control samples for each probe. ( B ) IHC staining of colonic tissue from VCMsh2T Hu mice. Images shown are a single field of view (original magnification, ×20). Scale bar: 200 μm. ( C ) A representative image of sequential immunofluorescence using Lunaphore COMET platform from VCMsh2T Hu colonic mucosa ( N = 3/group) stained with DAPI (red), E-cadherin (blue), CD8 (green), Ki67 (yellow), and CD163 (white) (original magnification, ×20). ( D ) Quantification of Comet data shown in C . ( E ) EZH2 knockdown in mouse organoids phenocopied similar results obtained with GSK503 inhibition of EZH2. Quantitative gene expression analysis results demonstrated significant changes in gene expression for Cdx 2 ( P = 0.0026), Dpp4 ( P = 0.022), Epcam ( P = 0.0003), Lgr5 ( P = 0.001), and Muc2 ( P = 0.0005). The mRNA levels of Krt20 and Vill were not significant. The graphed data are expressed as mean ± SEM. For all graphs, Student’s t test was used to determine significance. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Slides were then incubated in secondary antibody (ImmPRESS secondary antibody [HRP polymer] was used depending on the primary source; goat anti-rabbit IgG was used as EZH2 primary antibody [Vector Laboratories, MP-7451]; goat anti-mouse IgG was used as Ki-67 secondary antibody [Vector Laboratories, MP-7452]) for 1 hour at room temperature and then exposed to DAB (Vector Laboratories) for 1 minute followed by Mayer’s hematoxylin counterstain for 1 minute.

Techniques: Western Blot, Stripping Membranes, Control, Modification, Activity Assay, Immunohistochemistry, Immunofluorescence, Staining, Knockdown, Inhibition, Gene Expression

Journal: Scientific Reports

Article Title: EZH2 specifically regulates ISL1 during embryonic urinary tract formation

doi: 10.1038/s41598-024-74303-w

Figure Lengend Snippet: EZH2 enhance ISL1 expression through binding on Fragment 2. A) schematic reppresentation of the region tested for ChIP-qPCR in ISL1 (red segments) with distance relative to the 5’ of Fragment 2 in kb. B) ChIP-qPCR fold change representing EZH2 bound genomic DNA relative to mouse IgG bound DNA. Androgen receptor (ar) genomic DNA as positive control and actin b as negative control. All fold change except for actin b are significant over IgG control. C) Western blot against EZH2 shows a succefull siRNA knockdown of EZH2 compared to control. D) ISL1 qPCR with EZH2 siRNA dispalys a reduced ISL1 signal. E) Western blot on EZH2 with control vector and EZH2 overexpression plasmid confirms the efficency of the overexpression. F) Luciferase assay with EZH2 overexpression (red bars) and control (gray bars). As before, vector (pGL3) and Fragment 2 forward show only little expression in both cases while EZH2 overexpression enchances the reporter activitiy of the plasmid with flipped Fragment 2 significantly.

Article Snippet: The membrane was then blocked in 1 x TBS with 0.1% Tween20 and 5% milk powder solution for 2 h at room-temperature, cut horizontally to separate EZH2 and beta-actin and then incubated at 4oC with primary EZH2 antibody (ThermoFisher, Cat. No. #49-1043; 1 : 1.000) and anti-beta actin antibody (Sigma-Aldrich-A2228-RRID: AB_476697; 1 : 50.000).

Techniques: Expressing, Binding Assay, ChIP-qPCR, Positive Control, Negative Control, Control, Western Blot, Knockdown, Plasmid Preparation, Over Expression, Luciferase

Journal: Scientific Reports

Article Title: EZH2 specifically regulates ISL1 during embryonic urinary tract formation

doi: 10.1038/s41598-024-74303-w

Figure Lengend Snippet: Ezh2 mediates isl1 regulation with tissue specificity on the nephric region and causes defective nephric duct development. (A) isl1 in situ hybridization in WT, ezh2 +/- and KO larvae at 56 hpf. Red arrows indiacate the expression of isl1 in the nephric region that shows clear staining in the WT and ezh2 +/- lines and a strong reduction in the ezh2 KO zfl. Isl1 expression results almost not alterated in the brain and spinal chord of all genotypes. (B) Normalized isl1 qPCR in head-chopped embryo at 56 hpf shows a significantly reduced signal in the ezh2 KO line. (C) Normalized isl1 qPCR of whole zfl at 56 hpf shows no significant reduction of isl1 expression. (D) Isl1 immuno histochemistry (red cells) in Tg(wt1b: eGFP) line (in green) and double transgenic Tg(wt1b: eGFP) – ezh2 KO line. Left pannel indicates the location of the transversal paraffin section in reference of the whole zfl and nephric region. Blue circles indicate the sagittal nephric ducts; purple circle indicates the glomeruli region, white asterics the sagittal nephric ducts and the white plus the pancreas. Right pannels show the 3D co-localization of Isl1 protein (red) on the glomeruli and nephric ducts (green) in the WT (top) and ezh2 KO (lower) 56 hpf zfl. A clear absence of Isl1 signal locates to the glomeruli and nephric ducts of the ezh2 KO line compared to WT. (E) Nephric ducts of the ezh2 KO larvae display developmental defects and malformation at 3 dpf. Red arrows indicate the correct protrusion of the nephric duct in the WT and absence of GFP signal, togeather with dilated nephric ducts in the ezh2 KO.

Article Snippet: The membrane was then blocked in 1 x TBS with 0.1% Tween20 and 5% milk powder solution for 2 h at room-temperature, cut horizontally to separate EZH2 and beta-actin and then incubated at 4oC with primary EZH2 antibody (ThermoFisher, Cat. No. #49-1043; 1 : 1.000) and anti-beta actin antibody (Sigma-Aldrich-A2228-RRID: AB_476697; 1 : 50.000).

Techniques: In Situ Hybridization, Expressing, Staining, Immunohistochemistry, Transgenic Assay, Paraffin Section

Journal: Scientific Reports

Article Title: EZH2 specifically regulates ISL1 during embryonic urinary tract formation

doi: 10.1038/s41598-024-74303-w

Figure Lengend Snippet: Fragment 2 promoter regulates specifically ISL1 expression through EZH2 and disturbs the normal basal expression of ISL1-DT/ISL1 cluster. (A) Upper panel shows the genomic overview of ISL1 location with 115 bp upstream the reverse orientated ISL1-DT . Black stripe indicates the position of Fragment 2 and the arrow its active orientation. Lower panel displays chromatin interaction from sci-ATAC-seq3 for ISL1 in the fetal ureteric bud cells . Light blue bridge indicates the interaction of the genomic locus where Fragment 2 resides with the shared promoter region of ISL1 and ISL1-DT (light blue bridge). (B) Human adult RNA-seq shows the expression pattern of ISL1 and ISL1-DT in different tissues. These genes are generally expressed with higher counts for ISL1 and lower for ISL1-DT . (C) qPCR of ISL1 and ISL1-DT with control and EZH2 siRNA shows a reduced signal of ISL1 but unchanged ISL1-DT expression in HEK 293 cells. (D) Proposed molecular mechanism for the expression pattern of ISL1 and ISL1-DT mediated by EZH2. Top panel shows physiological condition, EZH2 binds to the Fragment 2 promoter and specifically enhances ISL1 expression (red arrows). In pathological condition, EZH2 does not bind to the Fragment 2 and the expression of ISL1 is reduced, but not the expression of ISL1-DT .

Article Snippet: The membrane was then blocked in 1 x TBS with 0.1% Tween20 and 5% milk powder solution for 2 h at room-temperature, cut horizontally to separate EZH2 and beta-actin and then incubated at 4oC with primary EZH2 antibody (ThermoFisher, Cat. No. #49-1043; 1 : 1.000) and anti-beta actin antibody (Sigma-Aldrich-A2228-RRID: AB_476697; 1 : 50.000).

Techniques: Expressing, RNA Sequencing, Control

Journal: Discover Oncology

Article Title: Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

doi: 10.1007/s12672-024-01056-3

Figure Lengend Snippet: Gene expression changes after IL-11Rα knockdown or IL-11 exogenous expression in the OS cells. a , Four shRNA sequences (G2, C1, H8, and H2) targeting IL-11Rα were stably expressed in KRIB cells. Gene expression was measured by microarray. Processed and log2-transformed gene expression data were compared to that of control cells. The heat map shows subtracted values for the gene probes in which values for shRNA sequences C1, H8, and H2 differed on average by at least twofold from those in the control cells. The color bar shows the fold-change difference from the values in the control cells. b , Quantitative reverse-transcription PCR analysis of the mRNA expression levels of the PRC2 complex members EZH2 , EED , and SUZ12 in SJSA1 (upper panel) and KRIB cells (lower panel). P—parental cells, E—empty vector, C1—IL-11Rα knockdown via shRNA C1, H8—IL-11Rα knockdown via shRNA H8, RQ—relative quantity. c Immunoblot of EZH2 and EED protein levels in SJSA1 and KRIB cells. β-actin served as a loading control. P—parental cells, E—empty vector, C1—IL-11Rα knockdown via shRNA C1, H8—IL-11Rα knockdown via shRNA H8, RQ—relative quantity.

Article Snippet: Primary antibodies against EZH2 (Cell Signaling Technology, 5246), EED (GeneTex, Inc., GTX628007), H3K27me3 (Millipore Sigma, 07–449), total histone (Abcam, ab1791) and β-actin (Cell Signaling Technology, 12620) were performed.

Techniques: Gene Expression, Knockdown, Expressing, shRNA, Stable Transfection, Microarray, Transformation Assay, Control, Reverse Transcription, Plasmid Preparation, Western Blot

Journal: Discover Oncology

Article Title: Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

doi: 10.1007/s12672-024-01056-3

Figure Lengend Snippet: Exogenous addition of human IL-11 to OS cells enhances PRC2 activity. Cells were treated with human recombinant IL-11 (hIL-11; 100, 200, and 300 ng) for 1 h and analyzed by a , qPCR for EZH2 , EED , and SUZ12 values, normalized to PPIA, and b , immunoblotting for H3K27 methylation status and expression of PRC2 complex member proteins (EZH2, SUZ12, and EED). Total histone and β-actin were used as loading controls. c Densitometry measurements of H3K27me3 levels normalized to total histone H3 for each dose of hIL-11. UT – untreated, **** P < 0.0001. d Kaplan–Meier curves showing probability of recurrence-free survival by extent of EZH2 staining. e – g The extent of positive EZH2 staining (percentage of positively stained nuclei) was scored on TMAs composed of 200 formalin-fixed, paraffin-embedded, decalcified human OS samples (141 primary and 59 metastatic). e Strong diffuse nuclear labeling. f Weak nuclear labeling. g Negative. Magnification 200X

Article Snippet: Primary antibodies against EZH2 (Cell Signaling Technology, 5246), EED (GeneTex, Inc., GTX628007), H3K27me3 (Millipore Sigma, 07–449), total histone (Abcam, ab1791) and β-actin (Cell Signaling Technology, 12620) were performed.

Techniques: Activity Assay, Recombinant, Western Blot, Methylation, Expressing, Staining, Formalin-fixed Paraffin-Embedded, Labeling

Journal: Discover Oncology

Article Title: Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

doi: 10.1007/s12672-024-01056-3

Figure Lengend Snippet: IL-11 activates EZH2 OS cells a Immunoprecipitation assay of EZH2 in CCH-OS-D and SJSA1 cells treated with human IL11 (200 or 400 ng) for 24 h. b CCH-OS-D cells were treated with human IL-11 for 48 h and then used for immunoblot analysis of phosphorylated EZH2-Y244. β-actin was used as loading control

Article Snippet: Primary antibodies against EZH2 (Cell Signaling Technology, 5246), EED (GeneTex, Inc., GTX628007), H3K27me3 (Millipore Sigma, 07–449), total histone (Abcam, ab1791) and β-actin (Cell Signaling Technology, 12620) were performed.

Techniques: Immunoprecipitation, Western Blot, Control

Journal: Discover Oncology

Article Title: Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

doi: 10.1007/s12672-024-01056-3

Figure Lengend Snippet: IL-11 mediates H3K27 trimethylation. a KRIB and SJSA1, EZH2shRNA and vector control cells were treated with human IL-11 for 48 h, Cells were harvested and used for immunoblot with H3K27 antibody. Total H3 was used as a control. b Immunoblot analysis of EZH2 knockdown and vector control in KRIB and SJSA1 cells. β-actin was used as a loading control

Article Snippet: Primary antibodies against EZH2 (Cell Signaling Technology, 5246), EED (GeneTex, Inc., GTX628007), H3K27me3 (Millipore Sigma, 07–449), total histone (Abcam, ab1791) and β-actin (Cell Signaling Technology, 12620) were performed.

Techniques: Plasmid Preparation, Control, Western Blot, Knockdown

Journal: Discover Oncology

Article Title: Targeting IL-11R/EZH2 signaling axis as a therapeutic strategy for osteosarcoma lung metastases

doi: 10.1007/s12672-024-01056-3

Figure Lengend Snippet: Inhibition of OS lung metastases by GSK126 in vivo. a Luciferase imaging of mice injected with SJSA1 cells comparing day 3, when primary tumors had developed, and day 21, after primary tumors had been removed by amputation but before treatment initiation. The bottom panels shows luciferase imaging and lung tumors for mice treated with control-, vehicle (cyclodextrin)-, and EZH2 inhibitor (EZH2i; GSK126) by day 48 after the start of the regimen. b Quantification of luciferase activity. c number of tumor nodules in control-treated versus GSK126-treated mice. d Survival curves for all mice. e Weight of lungs in control- and GSK126-treated mice. F Representative images of H&E staining of explanted lungs treated with Vehicle and GSK126. Scale bar: 100 μm

Article Snippet: Primary antibodies against EZH2 (Cell Signaling Technology, 5246), EED (GeneTex, Inc., GTX628007), H3K27me3 (Millipore Sigma, 07–449), total histone (Abcam, ab1791) and β-actin (Cell Signaling Technology, 12620) were performed.

Techniques: Inhibition, In Vivo, Luciferase, Imaging, Injection, Control, Activity Assay, Staining

Journal: Cell Death Discovery

Article Title: NCAPD3 exerts tumor-promoting effects in prostatic cancer via dual impact on miR-30a-5p by STAT3-MALAT1 and MYC

doi: 10.1038/s41420-024-01930-7

Figure Lengend Snippet: A Ten gene expression profiles of condensin complexes in three pairs of prostate cancer tissues and matched adjacent normal tissues were based on RNA-seq results. B qRT-PCR analysis of NCAPD3 mRNA levels in 20 pairs of PCa tissues and adjacent normal tissues. C The mRNA levels of NCAPD3 in PCa tissues and normal tissues were analyzed using GEPIA. D , E The protein levels of NCAPD3 in PCa tissues and adjacent normal tissues were detected by Western blot assay ( D ) and IHC staining ( E ). F The protein levels of NCAPD3 in prostate cancer cell lines (PC-3, DU145, 22Rv1, and LNCaP) and non-tumor prostate cell lines (WPMY1 and BPH1) were detected by Western blotting. G The protein expression of NCAPD3 and STAT3 was determined by western blot assay in the stable NCAPD3-overexpressing PC-3 cell line (PC-3-Lv-NCAPD3, abbreviated as PC-3-Lv-NC3) or its negative control cell line (PC-3-Lv-Control, abbreviated as PC-3-Lv-Ctrl), and the stable NCAPD3 knockdown 22Rv1 cells (22Rv1-Lv-shNCAPD3, abbreviated as 22Rv1-Lv-shNC3) or its negative control cell line (22Rv1-Lv-shControl, abbreviated as 22Rv1-Lv-shCtrl). H qRT-PCR analysis of MALAT1 expression in PC-3-Lv-NCAPD3/Control and 22Rv1-Lv-shNCAPD3/Control. I qRT-PCR analysis of MALAT1 expression in transiently transfected cells with STAT3 plasmid (STAT3) or empty vector (Ctrl) in PC-3 or STAT3 siRNA (siSTAT3) or negative control siRNA (siNC) in 22Rv1. J qRT-PCR analysis of MALAT1 expression in PC-3-Lv-NCAPD3/Control transiently transfected STAT3 siRNA (siSTAT3) or negative control siRNA (siNC), and in 22Rv1-Lv-shNCAPD3/Control transiently transfected STAT3 plasmid (STAT3) or empty vector (Ctrl). The Western blotting bands were semi-quantitatively analyzed using ImageJ and normalized to β-actin density. Scale bars: 200 and 100 μm, respectively. Normal represents adjacent normal tissues, and Tumor represents prostate cancer patient tissues. Values are means ± SE from n = 3 independent repetitions, * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 based on two-tailed Student’s t -test.

Article Snippet: Other primary antibodies for STAT3, AR, MYC, EZH2, AGO2, Ki67, β-actin and the secondary antibodies, including HRP Goat Anti-Mouse IgG and HRP Goat Anti-Rabbit IgG, were obtained from ABclonal Technology (Hubei, China).

Techniques: Gene Expression, RNA Sequencing, Quantitative RT-PCR, Western Blot, Immunohistochemistry, Expressing, Negative Control, Control, Knockdown, Transfection, Plasmid Preparation, Two Tailed Test

Journal: Cell Death Discovery

Article Title: NCAPD3 exerts tumor-promoting effects in prostatic cancer via dual impact on miR-30a-5p by STAT3-MALAT1 and MYC

doi: 10.1038/s41420-024-01930-7

Figure Lengend Snippet: A , B qRT-PCR analysis of miR-30a-5p expression in stable NCAPD3 overexpression or knockdown cells ( A ) and transiently transfected cells with NCAPD3 plasmid or siRNA ( B ). C , D Detection of miR-30a-5p labeled by Cy3 (red) by RNA FISH in stable NCAPD3 overexpression or knockdown cells ( C ) and transiently transfected cells with NCAPD3 plasmid or siRNA ( D ). E The expression efficiency of AR in PC-3 transiently transfected with AR plasmid was demonstrated by Western blot assay. F qRT-PCR analysis of AR, NCAPD3, and miR-30a-5p expression in PC-3 transiently transfected with AR plasmid. G , H 22Rv1 cells were transfected siAR or siNC and incubated with phenol-red-free media containing 10% CSS for 48 h, then treated with or without 10 nM Mib for another 24 h. Protein levels of AR, NCAPD3, and β-actin were checked by Western blotting ( G ). qRT-PCR analysis of miR-30a-5p expression was detected in cells ( H ). I qRT-PCR analysis of miR-30a-5p expression in PC-3, DU 145, 22Rv1, LNCaP, WPMY-1 and BPH-1. J , K Detection of miR-30a-5p expression in PCa tissues and adjacent normal tissues by qRT-PCR ( J ) and FISH ( K ). Scale bars: 100 μm. Normal represents adjacent normal tissues, Tumor represents prostate cancer patient tissues. Values are means ± SE from n = 3 independent repetitions, * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001 based on two-tailed Student’s t -test.

Article Snippet: Other primary antibodies for STAT3, AR, MYC, EZH2, AGO2, Ki67, β-actin and the secondary antibodies, including HRP Goat Anti-Mouse IgG and HRP Goat Anti-Rabbit IgG, were obtained from ABclonal Technology (Hubei, China).

Techniques: Quantitative RT-PCR, Expressing, Over Expression, Knockdown, Transfection, Plasmid Preparation, Labeling, Western Blot, Incubation, Two Tailed Test