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: Porphyromonas gingivalis promotes oral squamous cell carcinoma progression via the IL-6/EZH2/Snai2 axis

doi: 10.1038/s41598-026-41528-w

Figure Lengend Snippet: Exploration of the functions of EZH2 and Snai2 in the OSCC progressions. (A) EZH2 and Snai2 expression level in primary OSCC cells (POSCC) or normal oral epithelial cells as control (NC) before and after IL-6 treatment. (B) EZH2 and Snai2 mRNA transcription level in primary OSCC cells (POSCC) before and after IL-6 treatment. (C) Cell proliferation assay of the OSCC cell line, HSC-2, with different treatments targeting EZH2. EZH2-OE: EZH2 overexpression; EZH2-KD: EZH2 knockdown; EZH2-KD & ReEZH2: EZH2 knockdown cells with EZH2 recombinant protein rescuing management. (D) Cell migration assay of HSC-2 with different treatments targeting EZH2. The cell migration rate was monitored by RTCA analyzer and calculated as cell index. (E) mRNA transcription level of Snai2 in HSC-2 with different treatments targeting EZH2. (F) Cell proliferation assay of HSC-2 with different treatments targeting Snai2. Snai2-OE: Snai2 overexpression; Snai2-KD: Snai2 knockdown. (G) Cell migration assay of HSC-2 with different treatments targeting Snai2. Each experiment for RTCA analysis was performed with duplication of samples ( N = 2), while for other items was with triplication of samples ( N = 3) and the error bars were presented as SD result. The independent experiment was performed at least three times. For the statistic test using, results in panel B were analyzed with student’s t-test, in panel C, E and F were analyzed with One-way ANOVA method, and in D and G were with Two-way ANOVA method.

Article Snippet: Samples were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated with the EZH2 primary antibody (1: 1000, orb1939414, biorbyt, UK) and Snai2 primary antibody (1: 1000, orb197797, biorbyt, UK) overnight at 4 °C.

Techniques: Expressing, Control, Proliferation Assay, Over Expression, Knockdown, Recombinant, Cell Migration Assay, Migration

Journal: Scientific Reports

Article Title: Porphyromonas gingivalis promotes oral squamous cell carcinoma progression via the IL-6/EZH2/Snai2 axis

doi: 10.1038/s41598-026-41528-w

Figure Lengend Snippet: Determination of the effects of EZH2 regulated Snai2 signal on OSCC progression. (A) Wound healing images (left panel) and the quantification data (right panel) of HSC-2 from 0 h to 24 h, in different groups. EZH2-OE: EZH2 overexpression; SiSnai2: SiRNA treatment targeting Snai2. (B) Cell proliferation assay of HSC-2 with different treatments. (C) Cell migration assay of HSC-2 with different treatments. The cell migration rate was monitored by RTCA analyzer and calculated as cell index. (D) Cell proliferation assay of POSCC with different treatments. POSCC: primary OSCC cells without treatment; IL-6: primary OSCC cells with IL-6 stimulation; IL-6 & inEZH2: primary OSCC cells with EZH2 inhibitor treatment after IL-6 stimulation; IL-6 & SiSnai2: primary OSCC cells with Snai2 SiRNA treatment after IL-6 stimulation. (E) Cell migration assay of POSCC with different treatments. Each experiment for RTCA analysis was performed with duplication of samples ( N = 2), while for other items was with triplication of samples ( N = 3) and the error bars were presented as SD result. The independent experiment was performed at least three times. For the statistic test using, results in panel A, B and D were analyzed with One-way ANOVA method, and in C and E were with Two-way ANOVA method.

Article Snippet: Samples were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated with the EZH2 primary antibody (1: 1000, orb1939414, biorbyt, UK) and Snai2 primary antibody (1: 1000, orb197797, biorbyt, UK) overnight at 4 °C.

Techniques: Over Expression, Proliferation Assay, Cell Migration Assay, Migration

Journal: Scientific Reports

Article Title: Porphyromonas gingivalis promotes oral squamous cell carcinoma progression via the IL-6/EZH2/Snai2 axis

doi: 10.1038/s41598-026-41528-w

Figure Lengend Snippet: Bioinformatic analysis of relationships among Snai2, EMT, and P.g. infection. (A) GSEA enrichment of Snai2 in OSCC correlating with the terms from Biological Process (right panel), Molecular Functions (mid panel), and Cellular Structures (left panel). (B) Volcano plot of the DEGs in OSCC samples standardized to the normal samples. (C) The Kaplan–Meier (K-M) survival curve of OSCC patients with Snai2 expression. The x-axis and y-axis were pointing time and survival probability, respectively, and the high Snai2 expression group and low Snai2 expression group were illustrated as red line and blue line, respectively. D , E. Functional enrichment of the screened DEGs in KEGG database with the Benjamini & Hochberg analyzing method. Relevant signal pathways (D) and their relationships with IL-6 and Snai2 (E) were shown. F. PPI analysis of the DEGs corresponding proteins in the selected genes group from KEGG enrichment. The selected proteins were divided to two groups, the infection relevant proteins (orange) and the cancer metastasis relevant proteins (teal). The proteins IL-6, EZH2 and Snai2 were highlighted with green color.

Article Snippet: Samples were blocked with 5% non-fat milk in TBST for 1 h at room temperature and incubated with the EZH2 primary antibody (1: 1000, orb1939414, biorbyt, UK) and Snai2 primary antibody (1: 1000, orb197797, biorbyt, UK) overnight at 4 °C.

Techniques: Infection, Expressing, Functional Assay

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