Journal: Nature Communications

Article Title: Lack of SMARCB1 expression characterizes a subset of human and murine peripheral T-cell lymphomas

doi: 10.1038/s41467-024-52826-0

Figure Lengend Snippet: A UMAP plot showing 24 clusters of the integrated scRNA-seq dataset from two control spleen samples (WT) and two PTCL-NOS Smarcb1− tumor samples. B Relative abundance of different cell types in murine WT spleens (left), PTCL spleens (middle), and human tumors (right; NB: in order to ensure comparability, the stromal cells were removed before quantification). The pie charts in the lower part show the ratio between B-cells and myeloid cells. C Multiplex immunofluorescence (IF) images of FFPE sections of murine PTCL-NOS Smarcb1− and control spleen samples (WT: upper panels; tumor: lower panels). For better visualization, the white boxed areas ( a to f ) are enlarged (2.5x; scale bar = 100 µm). DAPI (gray) provides a nuclear counterstain, Ezh2 (yellow) defines malignant cells (Ezh2 hi ), B220 (blue) is used as a pan B-cell marker (B220 + ), and Ly6g (pink) as a marker for neutrophils (Ly6g + ). D Quantitative analysis of IF images from ( C ). Four representative regions of interest (ROIs; size: 1500 × 1500 µm) were selected and analyzed for mouse WT and Tumor samples. A Wilcoxon-Mann-Whitney test was calculated to determine if there are differences between WT and Tumor samples for all comparisons (* p = 0.0286). Boxplot settings: middle, median; lower hinge, 25% quantile; upper hinge, 75% quantile; upper/lower whisker, largest/smallest observation less/greater than or equal to upper/lower hinge ±1.5 * IQR. E The heatmap shows the overlap between cluster-specific DEG lists and the cancer hallmark metaprograms. F Signature plots of the programs Cycling, MYC, EMT and Stress in cells from WT (left) and tumor (right) samples. G A split violin plot (left/gray half: WT; right/black half: tumor) illustrates the increase in T-cell exhaustion features (Exhaust.) with a simultaneous decrease in NK cytotoxicity (Cytotox.) markers (e.g., Ncr1/NKp46) as well as infiltration of immunosuppressive myeloid cells in tumor versus WT samples. Source data of B and D are provided as a Source Data file. B Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

Article Snippet: For multiplexed immunofluorescence analysis, slices of PTCL-NOS Smarcb1− and corresponding murine control spleens were stained in the MACSima imaging system using antibodies against B220 (RA3-6B2, Miltenyi Biotec, APC, 1:50), Ly6G (1A8, Miltenyi Biotec, PE, 1:50) and EZH2 (REA907, Miltenyi Biotec, APC, 1:50).

Techniques: Control, Multiplex Assay, Immunofluorescence, Marker, MANN-WHITNEY, Whisker Assay

Journal: bioRxiv

Article Title: SUN2 mediates epigenetic remodeling to drive mechanotransduction during skin fibrosis

doi: 10.64898/2026.03.19.712957

Figure Lengend Snippet: (A-C) Images and quantification of Ezh2 immunofluorescence in WT and Sun2 KO dermal fibroblasts cultured on soft or stiff substrates. (A) Scale bar = 10 μ m. (B) Quantification of nuclear Ezh2 intensity. N = 2, n soft = 21–37, n stiff = 20–24. Significance was assessed using a two-tailed Mann–Whitney U test. (C) qPCR analysis of Ezh2 mRNA expression in WT or Sun2 KO fibroblasts on soft or stiff substrates. N = 3. Significance was assessed using a two-tailed Mann–Whitney U test. (D-E) Schematic and analysis of WT and Sun2 KO dermal fibroblasts plated on soft or stiff substrates for 24 h, followed by 24 h treatment with the Ezh2 inhibitor GSK343 or DMSO (Day 1), and collection for qPCR (Day 2). N = 3 independent experiments. Statistical significance was assessed using one-way ANOVA. ns, not significant; p < 0.05, * p < 0.01, ** p < 0.001, *** p < 0.0001. (F) Proposed model for SUN2–LINC–PRC2–dependent control of profibrotic gene expression in dermal fibroblasts. In quiescent WT fibroblasts, fibrotic genes are repressed. Upon mechanical stimulation of WT cells, cytoskeletal tension transmitted through LINC complexes promotes lamin stretching and altered Ezh2-driven epigenetic remodeling to activate transcription, repress transcriptional activators, and promote enhancer-licensing. In Sun2 KO dermal fibroblasts,loss of LINC complexes blunts this mechanotransduction despite cytoskeletal activation.

Article Snippet: Primary antibody diluted in blocking solution – SUN2, 1:200 (Abcam, AB124916), YAP, 1:200 (CST, 4912S), LaminA/C (Abcam, AB133256), and/or Ezh2, 1:200 (CST, 5246T) was incubated overnight at 4°C.

Techniques: Immunofluorescence, Cell Culture, Two Tailed Test, MANN-WHITNEY, Expressing, Control, Gene Expression, Activation Assay

Journal: Biochimica et biophysica acta

Article Title: Chromatin Dynamics Regulate Mesenchymal Stem Cell Lineage Specification and Differentiation to Osteogenesis

doi: 10.1016/j.bbagrm.2017.01.003

Figure Lengend Snippet: Expression patterns of genes Dmp1 from the up high group (A), Bmp3 from the up high group (B), Krt19 gene in the down group (C), and Igf2 gene from the bimodal group l_h_l_l (D) had distinct expression patterns during osteoblast differentiation. The enrichment dynamics of four selected histone marks (H3K4me3, H3K27ac, H3K27me3, and H3K36me3) are shown at the loci along with location of primers used in E. E) Occupancy of p300 and Ezh2 were evaluated by ChIP-PCR of at selected regions.

Article Snippet: Sheared chromatin was used for immunoprecipitation with Runx2 antibody (M-70, Santa Cruz), Ctcf (07-729, Millipore, Billerica, MA, USA), Smc1a (A300-055A, Bethyl Laboratories, Montgomery, TX, USA), H3K4me (ab8895, Abcam, Cambridge, MA, USA), H3K4me3 (ab1012, Abcam), H3K9me3 (Ab8898, Abcam), H3K9ac (39137, Active Motif), H3K27me3 (07-449, Millipore), H3K27ac (07-360, Millipore), H3K36me3 (ab9050, Abcam), ap300 (sc-585 X, Santa Cruz) or Ezh2 (A304-196A, Bethyl Laboratories) or immunoglobulin G (IgG) (12-370, Millipore) followed by purification using Protein-G Dynabeads (Invitrogen).

Techniques: Expressing

Journal: Molecular Nutrition & Food Research

Article Title: AMPK‐Dependent Epigenetic Regulation of Metabolism Mediates the Anti‐Cancer Action of Pterostilbene in Hepatocellular Carcinoma

doi: 10.1002/mnfr.70217

Figure Lengend Snippet: APMK‐dependent PTS epigenetic activation of metabolism‐associated genes is mediated by KDM6A demethylase, but not EZH2 methyltransferase or JMJD3 (KDM6B) demethylase. (A) The level of mRNA, as measured by qRT‐PCR, for EZH2 in liver tissues from the control group (choline‐sufficient diet, CSAA), HCC group (CDAA) and CDAA supplemented with PTS (CDAA+PTS). (B) Phosphorylated EZH2 at Thr311 (phospho‐EZH2, which indicates EZH2 activation) and total EZH2 levels as determined by western blot analysis and normalized by total protein load. The chart depicts the ratio of phospho‐EZH2 to total EZH2, as quantified based on band intensity values. (C and D) Occupancy of KDM6A (C) and JMJD3 (D) at the ALDH1L1 , BHMT , and DMGDH promoter regions in wild‐type HepG2 cells treated with 10 µM PTS for 24 hrs and in HepG2 cells pre‐treated with 10 µM of compound C (CC), an AMPK inhibitor, 24 h prior to PTS exposure. The occupancy was measured by chromatin immunoprecipitation (ChIP) followed by qPCR (qChIP) and compared to control—vehicle‐treated cells (ethanol for PTS; DMSO+ethanol for PTS+CC). Results represent mean ± SEM, * p < 0.05. (E) A scheme shows possible players in AMPK‐dependent decrease in histone H3 lysine 27 trimethylation (H3K27me3) in response to PTS. AMPK may act through the activation of KDM6 demethylases or via the inhibition of EZH2 methyltransferase. Created in BioRender. Boycott, C. (2025) https://BioRender.com/y60b739 .

Article Snippet: After blocking the membranes in EveryBlot Blocking buffer, membranes were incubated overnight at 4°C with the primary polyclonal antibody, anti‐AMPKα (1:1000; 5831, Cell Signaling Technology, Danvers, Massachusetts, USA), anti‐phospho‐AMPKα (Thr172) (1:1000; 2531, Cell Signaling Technology), anti‐EZH2 [Ezh2 (D2C9) XP Rabbit mAb, 1:1000, 5246, Cell Signaling Technology], and anti‐phospho‐EZH2 (Thr311) (1:1000; 27888, Cell Signaling Technology).

Techniques: Activation Assay, Quantitative RT-PCR, Control, Western Blot, Chromatin Immunoprecipitation, Inhibition

Journal: Nature Communications

Article Title: LncRNA-p21 alters the antiandrogen enzalutamide-induced prostate cancer neuroendocrine differentiation via modulating the EZH2/STAT3 signaling

doi: 10.1038/s41467-019-09784-9

Figure Lengend Snippet: EZH2, but not the PRC2 complex, is essential for Enz treatment induced NED. a , b Enz-increased NE markers were reduced after EZH2 knockdown. The C4-2 pLKO control and shEZH2 cells were treated with/without (w/o) 10 μM Enz for 4 days, and the NE markers detected by ( a ) western blot (WB) and ( b ) qPCR. c EZH2 knockdown can reverse the Enz-induced NED. The C4-2 pLKO and shEZH2 cells were treated w/o Enz for 7 days, then the cell morphology was observed under microscope, quantitations in lower panel. (Scale bar = 20 μm). d EZH2 inhibitors can inhibit the expression of NE markers. C4-2 cells were treated w/o Enz and EZH2 inhibitors (4 μM GSK126 and 1 μM Dznep) for 4 days, and the mRNAs of NE markers analyzed by qPCR. e Expression levels of NE markers in NE1.8 cells after knocking down EZH2. f C4-2 pWPI and pWPI-lncRNA-p21 cells were treated w/o 4 μM GSK126, and the NE markers levels detected by qPCR. g H3K27me3 levels in C4-2 cells after treating w/o Enz for 4 days. h H3K27me3 levels in C4-2 cells after DMSO, Enz and GSK126 treatment. i Co-IP assay to detect the interaction between EZH2, SUZ12 and EED in C4-2 cells w/o Enz treatment for 4 days. j GSEA of PRC2 and EZH2 pathway enrichment in C4-2 cells after Enz treatment. k Expression of PRC2 complex target genes in C4-2 cells after 4 days of Enz treatment. l The mRNA levels of NE markers in C4-2 pLKO control and C4-2 shEED cells after Enz treatment for 4 days. For b , d , f , k , the data are presented as mean ± SD, * p < 0.05, ** p < 0.005, by t- test

Article Snippet: The AKT (A01486), FOXA1 (EB05999), NSE (AP2780a), H3 (620–360), Methyl-K (SPC-158F), EED (A5371) and SUZ12 (AP20347b) antibodies were from OWL, Inc (San Diego, CA) and p-EZH2 antibody (IHC-00388) from Bethyl Laboratorys, Inc. (Montgomery, TX).

Techniques: Western Blot, Microscopy, Expressing, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: LncRNA-p21 alters the antiandrogen enzalutamide-induced prostate cancer neuroendocrine differentiation via modulating the EZH2/STAT3 signaling

doi: 10.1038/s41467-019-09784-9

Figure Lengend Snippet: EZH2 methylates STAT3 to promote NED after Enz treatment. a STAT3 methylation (Methyl-K) is increased in C4-2 cells treated with/without (w/o) 10 μM Enz for 4 days and the lysine methylation of STAT3 detected by anti-lysine methylation antibody. b EZH2 knockdown can reduce Enz increased STAT3 methylation. C4-2 pLKO vector and shEZH2 cells were treated w/o Enz for 5 days, and then STAT3 methylation and phosphorylation detected by WB. c EZH2 inhibitor can reduce Enz-Increased STAT3 methylation. The levels of STAT3 methylation and phosphorylation w/o Enz and GSK126 treatment were detected by WB. d GSEA of STAT3 pathway enrichment in C4-2 cells with Enz treatment. e STAT3 downstream genes’ expressions in C4-2 PLKO and shEZH2 cells after Enz treatment. f Co-IP assay to detect the interaction between EZH2 and STAT3 after Enz treatment. C4-2 cells were treated w/o Enz for 5 days. EZH2 antibody was used to pull-down the EZH2 complex, and the STAT3 level detected by WB. g Confocal microscopic images of co-localization of EZH2 and STAT3 after Enz or DMSO treatment for 4 days. The co-localization of STAT3 and EZH2 was analyzed by Pearsons coefficient (Scale bar = 2 μm). Quantitation on the right. h WB analysis of EZH2 p-S21 in C4-2 cells w/o Enz treatment daily for 4 days. i STAT3 inhibitor, S3I-201 (S3I), can reduce the expression of Enz-induced NE markers. C4-2 cells were treated w/o Enz and S3I for 6 days, and the NE markers determined by WB. j The p-EZH2, EZH2, p-STAT3 and STAT3 levels in NE1.8 and LNCaP cells were analyzed by WB. For e , g , the data are presented as mean ± SD, * p < 0.05, ** p < 0.005 by t- test

Article Snippet: The AKT (A01486), FOXA1 (EB05999), NSE (AP2780a), H3 (620–360), Methyl-K (SPC-158F), EED (A5371) and SUZ12 (AP20347b) antibodies were from OWL, Inc (San Diego, CA) and p-EZH2 antibody (IHC-00388) from Bethyl Laboratorys, Inc. (Montgomery, TX).

Techniques: Methylation, Plasmid Preparation, Co-Immunoprecipitation Assay, Quantitation Assay, Expressing

Journal: Nature Communications

Article Title: LncRNA-p21 alters the antiandrogen enzalutamide-induced prostate cancer neuroendocrine differentiation via modulating the EZH2/STAT3 signaling

doi: 10.1038/s41467-019-09784-9

Figure Lengend Snippet: The lncRNA-p21 disrupts the PRC2 complex and promotes EZH2 to methylate STAT3. a The qPCR analysis of PRC2 target gene expressions in C4-2 cells after overexpression of lncRNA-p21. b EZH2 protein was immunoprecipitated in C4-2 pWPI and pWPI-lncRNA-p21 cells. The co-immunoprecipitated SUZ12 levels were detected by WB. The SYP and H3K27Me3 levels in input samples were also detected by WB. c The WB analysis of STAT3 methylation and phosphorylation with/without (w/o) overexpression of lncRNA-p21 in C4-2 cells. d The qPCR analysis of STAT3 target genes expressions in C4-2 cells w/o overexpression of lncRNA-p21. e The lncRNA-p21 knockdown can reduce Enz-induced STAT3 methylation. C4-2 pLKO and 2 different sh-lncRNA-p21 transfected cells were treated w/o Enz for 4 days. The STAT3 methylation was detected by WB. f Co-IP assay to detect the interaction between EZH2 and STAT3 w/o overexpression of lncRNA-p21 in C4-2 cells. g Confocal microscopic images of co-localization of EZH2 and STAT3 w/o Enz treatment in C4-2 pLKO and sh-lncRNA-p21 cells. The co-localization of EZH2 and STAT3 was analyzed by Pearsons coefficient (Scale bar = 2 μm). Quantitation on the right. For a , d , g , data are presented as mean ± SD, * p < 0.05, ** p < 0.005, N.S. not significant by t- test

Article Snippet: The AKT (A01486), FOXA1 (EB05999), NSE (AP2780a), H3 (620–360), Methyl-K (SPC-158F), EED (A5371) and SUZ12 (AP20347b) antibodies were from OWL, Inc (San Diego, CA) and p-EZH2 antibody (IHC-00388) from Bethyl Laboratorys, Inc. (Montgomery, TX).

Techniques: Over Expression, Immunoprecipitation, Methylation, Transfection, Co-Immunoprecipitation Assay, Quantitation Assay

Journal: Nature Communications

Article Title: LncRNA-p21 alters the antiandrogen enzalutamide-induced prostate cancer neuroendocrine differentiation via modulating the EZH2/STAT3 signaling

doi: 10.1038/s41467-019-09784-9

Figure Lengend Snippet: The lncRNA-p21 competes with Hotair to interact with EZH2 and promotes phosphorylation of EZH2 at S21. a , b RNA-IP was performed to detect the lncRNA-p21 interaction with EZH2 by using lncRNA-p21 anti-sense probe vs sense probe in ( a ) C4-2 cells and in ( b ) Enz-treated C4-2 cells. c RNA in situ hybridization and immunofluorescent staining to detect the co-localization of lncRNA-p21 and EZH2 in C4-2 cells w/o Enz treatment (Scale bar = 2 μm). d In C4-2 cells, the EZH2 was pulled down by EZH2 antibody and the levels of lncRNA-p21 and Hotair were detected by qPCR. e , f RNA-IP assay to detect the interaction between Hotair and EZH2 after treating w/o Enz ( e ) and w/o overexpression of lncRNA-p21. f , g STAT3 methylation levels were detected by WB in C4-2 pLKO and sh-EED cells after 4 days w/o Enz treatment. h EZH2 p-S21 levels were detected in C4-2 PWPI and PWPI-lncRNA-p21 cells. i EZH2 p-S21 level was detected in C4-2 pLKO and sh-lncRNA-p21 cells w/o Enz treatment. j The Co-IP assay to detect the AKT and EZH2 interaction in C4-2 pWPI and C4-2 pWPI-lncRNA-p21 cells. k C4-2 pLKO and sh-lncRNA-p21 cells were treated w/o Enz for 4 days and then EZH2 was immunoprecipitated. The AKT and EZH2 interaction was analyzed by WB. l C4-2 cells were treated w/o Enz, and then the lncRNA-p21 was pulled down. The interaction of AKT and EZH2 with lncRNA-p21 was analyzed by WB. For d , the data are presented as mean ± SD, * p < 0.05, ** p < 0.005, by ANOVA

Article Snippet: The AKT (A01486), FOXA1 (EB05999), NSE (AP2780a), H3 (620–360), Methyl-K (SPC-158F), EED (A5371) and SUZ12 (AP20347b) antibodies were from OWL, Inc (San Diego, CA) and p-EZH2 antibody (IHC-00388) from Bethyl Laboratorys, Inc. (Montgomery, TX).

Techniques: RNA In Situ Hybridization, Staining, Over Expression, Methylation, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: Nature Communications

Article Title: LncRNA-p21 alters the antiandrogen enzalutamide-induced prostate cancer neuroendocrine differentiation via modulating the EZH2/STAT3 signaling

doi: 10.1038/s41467-019-09784-9

Figure Lengend Snippet: LncRNA-p21/EZH2/STAT3 signal is activated in PDX mice model after Enz treatment and human NEPC samples. a The PCa PDX samples were subcutaneously implanted into SCID mice and 5 mice/group received different i.p. treatments (1. DMSO; 2. Enz; 3. Dznep; 4. Dznep + Enz) every other day, and tumor sizes were measured after different treatments. After 10 treatments, the mice were kept for another 2 days and then were sacrificed.The PDX tumors were collected, and the relative tumor growth rates of different treatments were compared. b The IHC staining to identify the level of NE markers indicated in different groups (Scale bar = 20 μm). c Three tumor samples were randomly picked up from each group, the tissues were lysed and then STAT3 methylation (methyl-k) was detected by WB. d The lncRNA-p21 levels in control group and Enz-treated group were detected by qPCR. e The correlation of lncRNA-p21 expression and p-EZH2 level in human PCa samples ( n = 80). f Representative images of the immunohistochemistry staining of SYP, ChgA, p-EZH2 and p-STAT3 in human CRPC and SCC samples. (Scale bar = 20 μm). g The quantification of p-EZH2 (upper) and p-STAT3 (lower) levels in human CRPC and PCa-SCC samples. For a , e , g , the data are presented as mean ± SD, ** p < 0.005, by t test for two groups or ANOVA for more than two groups

Article Snippet: The AKT (A01486), FOXA1 (EB05999), NSE (AP2780a), H3 (620–360), Methyl-K (SPC-158F), EED (A5371) and SUZ12 (AP20347b) antibodies were from OWL, Inc (San Diego, CA) and p-EZH2 antibody (IHC-00388) from Bethyl Laboratorys, Inc. (Montgomery, TX).

Techniques: Immunohistochemistry, Methylation, Expressing, Staining

Journal: Nature Communications

Article Title: LncRNA-p21 alters the antiandrogen enzalutamide-induced prostate cancer neuroendocrine differentiation via modulating the EZH2/STAT3 signaling

doi: 10.1038/s41467-019-09784-9

Figure Lengend Snippet: Schematic depiction of lncRNA-p21/EZH2/STAT3 pathway. Enz treatment induces lncRNA-p21 expression via regulating the AR binding to the lncRNA-p21 promoter in PCa cells. Highly expressed lncRNA-p21 competes with Hotair to interact with EZH2 and disrupt PRC2 complex (SUZ12 and EED). At the same time, lncRNA-p21 promotes AKT phosphorylated EZH2 on Serine 21 and enhances the STAT3 methylation by EZH2. The methylated STAT3 was activated and promotes the NED

Article Snippet: The AKT (A01486), FOXA1 (EB05999), NSE (AP2780a), H3 (620–360), Methyl-K (SPC-158F), EED (A5371) and SUZ12 (AP20347b) antibodies were from OWL, Inc (San Diego, CA) and p-EZH2 antibody (IHC-00388) from Bethyl Laboratorys, Inc. (Montgomery, TX).

Techniques: Expressing, Binding Assay, Methylation

Journal: Immunobiology

Article Title: Transcription suppression of GABARAP mediated by lncRNA XIST-EZH2 interaction triggers caspase-11-dependent inflammatory injury in ulcerative colitis.

doi: 10.1016/j.imbio.2024.152796

Figure Lengend Snippet: Fig. 1. XIST is highly expressed in DSS-treated mice and is a possible gene correlated with inflammation. A, LncRNAs that can interact with EZH2 predicted using the RNA Inter tool; B, subcellular localization of XIST in cells predicted in the LncATLAS system. C57BL/6N mice were treated with 3 % DSS for 7 d or not. C, XIST expression in the colon tissue of mice examined by RT-qPCR (n = 6); D, body weight of mice in each group (n = 6); E, the length of colon of mice in each group (n = 6); F, DAI of mice in each group (n = 6); G, mucosal injury and inflammatory infiltration in the mouse colon determined by HE staining (n = 6); H, concentrations of inflammatory cytokines TNF-α, IL-1β, and IL-18 in the mouse colon tissue examined using ELISA kits (n = 6); I, protein levels of occludin, ASC, and cleaved caspase-1 in the mouse colon tissue determined by WB analysis (n = 6); J, concentrations of caspase-1 and caspase-11 in the mouse colon tissue determined using ELISA kits (n = 6). Differences were analyzed by the unpaired t test (C, E, H, and J) or two-way ANOVA (D, F, and I). * p < 0.05 vs. the Control group.

Article Snippet: These membranes were then probed with the antibodies of GABARAP (1:1000, GTX31541, GeneTex Inc., San Antonio, TX, USA), occludin (1:1000, GTX114949, GeneTex), ASC (1:1000, ab307560, Abcam Inc., Cambridge, MA, USA), EZH2 (1:2000, NBP2-17087; Novus Biologicals, Littleton, CA, USA), caspase-11 (1:1000, 21741–1-AP, Proteintech Group, Inc, Wuhan, Hubei, China), cleaved caspase-1 (1:1000, #89332, Cell Signaling Technology, Beverly, MA, USA) and β-actin (1:2000, GTX109639, GeneTex) overnight at 4°C.

Techniques: Expressing, Quantitative RT-PCR, Staining, Enzyme-linked Immunosorbent Assay, Control

Journal: Immunobiology

Article Title: Transcription suppression of GABARAP mediated by lncRNA XIST-EZH2 interaction triggers caspase-11-dependent inflammatory injury in ulcerative colitis.

doi: 10.1016/j.imbio.2024.152796

Figure Lengend Snippet: Fig. 4. XIST recruits EZH2 to regulate GABARAP expression. A, genes exhibiting a significant negative correlation with EZH2 in TCGA-COAD samples in the UALCAN system; B, a negative correlation between GABARAP and EZH2 in in normal colon samples in the GEPIA system; C, the existence of H3K27me3 modifications near the GABARAP promoter analyzed using the mouse H3K27me3 ChIP-seq sample in the ENCODE system; D, GABARAP mRNA expression in mouse colonic mucosal epithelial cells (Procell) after different treatments (PBS/DSS + LPS/KD-NC/KD-XIST) examined by RT-qPCR (n = 3); E, binding relationship between XIST and EZH2 in cells examined by RIP assay (n = 3); F-G, binding between EZH2 (F) and H3K27me3 (G) and the GABARAP promoter in cells examined by ChIP assay (n = 3); H, mRNA and protein levels of EZH2 in mice after different treatments (drinking water/DSS/KD-NC/KD-XIST transfection) determined by RT-qPCR and WB analysis, respectively (n = 6); I mRNA and protein levels of EZH2 in mouse colonic mucosal epithelial cells (Procell) after different treatments (PBS/DSS + LPS/KD-NC/KD- XIST) determined by RT-qPCR and WB analysis, respectively (n = 3); J-K, binding between H3K27me3 (J) and EZH2 (K) and the GABARAP promoter in cells after different treatments (PBS/DSS + LPS/KD-NC/KD-XIST) determined by ChIP assays (n = 3); L-M, mRNA (L) and protein (M) levels of GABARAP in cells transfected with KD-NC/KD-EZH2 determined by RT-qPCR or WB analysis (n = 3); N, binding between H3K27me3 and the GABARAP promoter in cells transfected with KD-NC/ KD-EZH2 examined by ChIP assay (n = 3). Differences were analyzed by the unpaired t test (E, F, G, L, M, and N) or one-way ANOVA (D, H, I, J, and K). * p < 0.05 vs. the IgG/Control/Normal group; # p < 0.05 vs. the KD-NC group.

Article Snippet: These membranes were then probed with the antibodies of GABARAP (1:1000, GTX31541, GeneTex Inc., San Antonio, TX, USA), occludin (1:1000, GTX114949, GeneTex), ASC (1:1000, ab307560, Abcam Inc., Cambridge, MA, USA), EZH2 (1:2000, NBP2-17087; Novus Biologicals, Littleton, CA, USA), caspase-11 (1:1000, 21741–1-AP, Proteintech Group, Inc, Wuhan, Hubei, China), cleaved caspase-1 (1:1000, #89332, Cell Signaling Technology, Beverly, MA, USA) and β-actin (1:2000, GTX109639, GeneTex) overnight at 4°C.

Techniques: Expressing, ChIP-sequencing, Quantitative RT-PCR, Binding Assay, Transfection, Control

Journal: Immunobiology

Article Title: Transcription suppression of GABARAP mediated by lncRNA XIST-EZH2 interaction triggers caspase-11-dependent inflammatory injury in ulcerative colitis.

doi: 10.1016/j.imbio.2024.152796

Figure Lengend Snippet: Fig. 5. Upregulation of EZH2 or knockdown of GABARAP aggravates inflammatory injury in mice with UC. A, GABARAP mRNA expression in the colon tissue of mice following different treatments (Drinking water/3% DSS/KD-NC/KD-XIST) examined by RT-qPCR (n = 6). UC mice transfected with KD-XIST were further administered KD-GABARAP/KD-NC or oe-EZH2/oe-NC. B, mRNA expression of EZH2 and GABARAP in the mouse colon tissues examined by RT-qPCR (n = 6); C, copies of viral particles in mouse colon tissues determined by qPCR analysis (n = 6); D, protein levels of occludin, ASC, and cleaved caspase-1 in the mouse colon tissue determined by WB analysis (n = 6); E, concentrations of caspase-1 and caspase-11 in the mouse colon tissue determined using ELISA kits (n = 6); F, body weight of mice in each group (n = 6); G, the length of colon of mice in each group (n = 6); H, DAI of mice in each group (n = 6); I, mucosal injury and inflammatory infiltration in the mouse colon determined by HE staining (n = 6); J, concentrations of TNF-α, IL-1β, and IL-18 in the mouse colon tissue examined using ELISA kits (n = 6). Differences were analyzed by the one-way (A, C, E, G, and J) or two-way (B, D, F, and H) ANOVA. * p < 0.05 vs. the KD-XIST + KD-NC group; & p < 0.05 vs. the KD-XIST + oe-NC group; @ p < 0.05 vs. the Control group; # p < 0.05 vs. the KD-NC group.

Article Snippet: These membranes were then probed with the antibodies of GABARAP (1:1000, GTX31541, GeneTex Inc., San Antonio, TX, USA), occludin (1:1000, GTX114949, GeneTex), ASC (1:1000, ab307560, Abcam Inc., Cambridge, MA, USA), EZH2 (1:2000, NBP2-17087; Novus Biologicals, Littleton, CA, USA), caspase-11 (1:1000, 21741–1-AP, Proteintech Group, Inc, Wuhan, Hubei, China), cleaved caspase-1 (1:1000, #89332, Cell Signaling Technology, Beverly, MA, USA) and β-actin (1:2000, GTX109639, GeneTex) overnight at 4°C.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, Transfection, Enzyme-linked Immunosorbent Assay, Staining, Control

Journal: Immunobiology

Article Title: Transcription suppression of GABARAP mediated by lncRNA XIST-EZH2 interaction triggers caspase-11-dependent inflammatory injury in ulcerative colitis.

doi: 10.1016/j.imbio.2024.152796

Figure Lengend Snippet: Fig. 6. Upregulation of EZH2 or knockdown of GABARAP increases inflammatory injury in mouse colonic mucosal epithelial cells. Mouse colonic mucosal epithelial cells (Procell) were transfected with KD-NC/KD-XIST and the additional KD-GABARAP/KD-NC or oe-EZH2/oe-NC, followed by DSS and LPS challenge. A, GABARAP and EZH2 mRNA expression in cells examined by RT-qPCR (n = 3); B, viability of cells examined by MTT assay (n = 3); C, apoptosis of cells examined by TUNEL assay (n = 3); D-F, concentrations of TNF-α (D), IL-1β (E), and IL-18 (F) in cells examined using ELISA kits (n = 3); G, concentrations of caspase-1 and caspase-11 in cells examined using ELISA kits (n = 3); H, protein level of cleaved-caspase-1 in cells determined by WB analysis (n = 3). Differences were analyzed by the one-way (B-H) or two-way (A) ANOVA. * p < 0.05 vs. the KD-XIST + KD-NC group; # p < 0.05 vs. the KD-XIST + oe-NC group.

Article Snippet: These membranes were then probed with the antibodies of GABARAP (1:1000, GTX31541, GeneTex Inc., San Antonio, TX, USA), occludin (1:1000, GTX114949, GeneTex), ASC (1:1000, ab307560, Abcam Inc., Cambridge, MA, USA), EZH2 (1:2000, NBP2-17087; Novus Biologicals, Littleton, CA, USA), caspase-11 (1:1000, 21741–1-AP, Proteintech Group, Inc, Wuhan, Hubei, China), cleaved caspase-1 (1:1000, #89332, Cell Signaling Technology, Beverly, MA, USA) and β-actin (1:2000, GTX109639, GeneTex) overnight at 4°C.

Techniques: Knockdown, Transfection, Expressing, Quantitative RT-PCR, MTT Assay, TUNEL Assay, Enzyme-linked Immunosorbent Assay

Journal: Immunobiology

Article Title: Transcription suppression of GABARAP mediated by lncRNA XIST-EZH2 interaction triggers caspase-11-dependent inflammatory injury in ulcerative colitis.

doi: 10.1016/j.imbio.2024.152796

Figure Lengend Snippet: Fig. 8. Graphical abstract. LncRNA XIST is upregulated in UC models. It recruits EZH2, which suppresses GABARAP transcription by binding to its promoter, consequently activating the caspase-11-dependent inflammatory cascade and UC development.

Article Snippet: These membranes were then probed with the antibodies of GABARAP (1:1000, GTX31541, GeneTex Inc., San Antonio, TX, USA), occludin (1:1000, GTX114949, GeneTex), ASC (1:1000, ab307560, Abcam Inc., Cambridge, MA, USA), EZH2 (1:2000, NBP2-17087; Novus Biologicals, Littleton, CA, USA), caspase-11 (1:1000, 21741–1-AP, Proteintech Group, Inc, Wuhan, Hubei, China), cleaved caspase-1 (1:1000, #89332, Cell Signaling Technology, Beverly, MA, USA) and β-actin (1:2000, GTX109639, GeneTex) overnight at 4°C.

Techniques: Binding Assay

Journal: Oncology Reports

Article Title: MicroRNA-218 promotes cisplatin resistance in oral cancer via the PPP2R5A/Wnt signaling pathway

doi: 10.3892/or.2017.5899

Figure Lengend Snippet: Suppression of miR-218 promotes cisplatin sensitivity in resistant oral cancer cells. (A) The tumor cells were transfected with anti-miR-218 or its control (anti-NC), and total RNA was extracted for qRT-PCR. (B and C) UM1 and Cal27 cells transfected with anti-miR-218 or its control were treated with cisplatin (2.5 µg/ml). The cell viability was assayed using the CCK-8 method. (D and E) Apoptosis was quantitated by flow cytometry. (F) The protein expression levels of chemoresistance-related proteins, including EZH2, ABCG2, MRP1, P-glycoprotein and TopoIIβ, were determined by western blotting. β-tubulin served as a loading control. (G) The expression levels of the chemoresistance-related proteins were quantitated. Data represent the mean ± SD of 3 independent experiments; *P<0.05, **P<0.01.

Article Snippet: The antibody against EZH2 was purchased from OriGene (Rockville, MD, USA).

Techniques: Transfection, Control, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry, Expressing, Western Blot