Journal: bioRxiv

Article Title: Triptolide ameliorates renal tubulointerstitial fibrosis through EZH2

doi: 10.1101/2023.01.29.526092

Figure Lengend Snippet: A and B . The expression of EZH2 in sham or UUO kidneys was analyzed by Western blotting and further quantified. C and D . HK2 human renal epithelial cells were starved overnight, then stimulated with TGF-β and treated with various concentrations of triptolide for 48h. The expression of EZH2 was analyzed by Western blotting and further quantified. One representative of at least three independent experiments is shown. Data represents the mean◻±◻SD. NS represents not significant. *** p ◻<◻0.001.

Article Snippet: Unspecific binding on the PVDF membrane was blocked by incubation with the blocking buffer (5% non-fat milk, 20mM Tris-HCl, 150mM NaCl, PH=8.0, 0.01%Tween 20) for 1 hour at room temperature, which was followed by incubation with anti-fibronectin (1:1000, ab23750, Abcam), anti-Collagen I (1:500, sc-293182, Santa Cruz), anti-α-SMA (1:1000, ET1607-53, HUABIO), anti-EZH2 (1:1000, 5246S, Cell Signaling Technology) antibody, anti-N-cadherin (1:1000, sc-59887, Santa Cruz), anti-Vimentin (1:1000, R1308-6, HUABIO), anti-Snail(1:1000, A11794, ABclonal), anti-pSmad3 (1:1000, ET1609-41, HUABIO), anti-GAPDH (1:5000, 60004-1-lg, Proteintech) antibodies overnight at 4◻.

Techniques: Expressing, Western Blot

Journal: bioRxiv

Article Title: Triptolide ameliorates renal tubulointerstitial fibrosis through EZH2

doi: 10.1101/2023.01.29.526092

Figure Lengend Snippet: HK2 human renal epithelial cells were starved overnight and followed by stimulation with TGF-β and treatment with 20 μM of 3-DZNeP and 100nM of triptolide for 48h. Cell lysates were extracted. A and B . The expression of EZH2 was analyzed by Western blotting and further quantified. C-E . The expression of fibronectin and collagen I were analyzed by Western blotting and then quantified. F and G . The expression of N-cadherin, vimentin and snail were analyzed by Western blotting and then quantified. H and I . The expression of phosphorylated Smad3 (pSmad3) was analyzed by Western blotting and then quantified. One representative of at least three independent experiments is shown. Data represents the mean◻±◻SD. NS represents not significant.* p ◻<◻0.05. ** p ◻<◻0.01. *** p ◻<◻0.001.

Article Snippet: Unspecific binding on the PVDF membrane was blocked by incubation with the blocking buffer (5% non-fat milk, 20mM Tris-HCl, 150mM NaCl, PH=8.0, 0.01%Tween 20) for 1 hour at room temperature, which was followed by incubation with anti-fibronectin (1:1000, ab23750, Abcam), anti-Collagen I (1:500, sc-293182, Santa Cruz), anti-α-SMA (1:1000, ET1607-53, HUABIO), anti-EZH2 (1:1000, 5246S, Cell Signaling Technology) antibody, anti-N-cadherin (1:1000, sc-59887, Santa Cruz), anti-Vimentin (1:1000, R1308-6, HUABIO), anti-Snail(1:1000, A11794, ABclonal), anti-pSmad3 (1:1000, ET1609-41, HUABIO), anti-GAPDH (1:5000, 60004-1-lg, Proteintech) antibodies overnight at 4◻.

Techniques: Expressing, Western Blot

Journal: PLOS Biology

Article Title: Loss of the m6A methyltransferase METTL3 in monocyte-derived macrophages ameliorates Alzheimer’s disease pathology in mice

doi: 10.1371/journal.pbio.3002017

Figure Lengend Snippet: (A) ChIP analysis of Dnmt3a at positions 5 kilobases (kb) (up5k), 4 kb (up4k), 3 kb (up3k), 2 kb (up2k), or 1 kb (up1k) upstream of the transcriptional start site of Atat1 in BMDMs. (B) ChIP analysis of H3K27me3 or EZH2 at the Atat1 promoter in the WT or KO BMDMs. (C) ChIP analysis of H3K27me3 or EZH2 at the Atat1 promoter in the DNMT3A knockdown BMDMs. (D) The expression of DNMT3A, EZH2, and ATAT1 was measured by qRT-PCR and western blot in the BMDMs transfected with NC, Dnmt3a , or Dnmt3a and Ezh2 siRNAs. All data in the figure are shown as the mean ± SD. P < 0.05 (*). Underlying data can be found in . BMDM, bone marrow-derived macrophage; ChIP, chromatin immunoprecipitation; KO, knockout; NC, negative control; qRT-PCR, quantitative reverse transcription PCR; WT, wild type.

Article Snippet: Primary antibodies were diluted in TBST containing 5% BSA and used at the indicated concentrations: rabbit anti-acetyl-α-tubulin (1:1,000, 5335, Cell Signaling Technology), rabbit anti-EZH2 (1:1,000, 5246, Cell Signaling Technology), rabbit anti-H3K27me3 (1:1,000, 9733, Cell Signaling Technology), rabbit anti-DNMT3A (1:1,000, ab2850, Abcam), rabbit anti-YTHDF1 (1:1,000, 17479-1-AP, 18810, Proteintech), rabbit anti-METTL3 (1:1,000, ab18810, Abcam), and mouse anti-β-actin (1:5,000, Sigma).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Derivative Assay, Chromatin Immunoprecipitation, Knock-Out, Negative Control

Journal: Cancers

Article Title: Evaluation of Tazemetostat as a Therapeutically Relevant Substance in Biliary Tract Cancer

doi: 10.3390/cancers15051569

Figure Lengend Snippet: Applied antibodies used in the immunohistochemical staining of three used BTC cell lines KKU-055, NOZ and OCUG-1.

Article Snippet: EZH2 , Cell Signaling , 5246S , D2C9 , High pH , rtu , Ultraview , Ventana.

Techniques: Immunohistochemical staining, Staining, Incubation

Journal: Cancers

Article Title: Evaluation of Tazemetostat as a Therapeutically Relevant Substance in Biliary Tract Cancer

doi: 10.3390/cancers15051569

Figure Lengend Snippet: Western Blot and mRNA expression analysis of EZH2 in KKU-055 and NOZ following tazemetostat treatment. ( A ) EZH2 mRNA expression and fold regulation after incubation with tazemetostat for 96 h in KKU-055 and NOZ cells; ( B ) EZH2 protein expression and fold regulation after incubation with tazemetostat for 96 h in KKU-055 and NOZ cells; ( C ) representative Western Blot images of H3 and EZH2 after the incubation of tazemetostat for 96 h in BTC cell lines KKU-055 and NOZ. UTC = untreated control. The uncropped blots are shown in .

Article Snippet: EZH2 , Cell Signaling , 5246S , D2C9 , High pH , rtu , Ultraview , Ventana.

Techniques: Western Blot, Expressing, Incubation

Journal: Cancers

Article Title: Evaluation of Tazemetostat as a Therapeutically Relevant Substance in Biliary Tract Cancer

doi: 10.3390/cancers15051569

Figure Lengend Snippet: mRNA expression analysis of EZH2-associated genes and Western Blot analysis of FBP1 in KKU-055 and NOZ following tazemetostat treatment. ( A ) Fold regulation of 21 genes in KKU-055 and NOZ after treatment with tazemetostat for 96 h (+2 and −2 = significant change). ( B ) FBP1 protein expression and fold regulation after incubation with tazemetostat for 96 h in KKU-055 and NOZ cells. ( C ) Representative Western Blot images of H3 and FBP1 after incubation of tazemetostat for 96 h in BTC cell lines KKU-055, NOZ. * = significant p < 0.05, UTC = untreated control. The uncropped blots are shown in .

Article Snippet: EZH2 , Cell Signaling , 5246S , D2C9 , High pH , rtu , Ultraview , Ventana.

Techniques: Expressing, Western Blot, Incubation

Journal: Cancers

Article Title: Evaluation of Tazemetostat as a Therapeutically Relevant Substance in Biliary Tract Cancer

doi: 10.3390/cancers15051569

Figure Lengend Snippet: EZH2 mutation status of biliary tract cancer cell lines, where Y641S represents a gain-of-function mutation. n.d.: not defined.

Article Snippet: EZH2 , Cell Signaling , 5246S , D2C9 , High pH , rtu , Ultraview , Ventana.

Techniques: Mutagenesis

Journal: Journal for Immunotherapy of Cancer

Article Title: MUC1-C is a master regulator of MICA/B NKG2D ligand and exosome secretion in human cancer cells

doi: 10.1136/jitc-2022-006238

Figure Lengend Snippet: MUC1-C represses MICA/B expression by driving EZH2-mediated H3K27 and DNMT-mediated DNA methylation. (A) RKO/CshRNA and RKO/NF-κBshRNA cells were analyzed for NF-κB, MICA and MICB mRNA levels by qRT-PCR. The results (mean±SD of four determinations) are expressed as relative mRNA levels compared with that obtained for vehicle-treated cells (assigned a value of 1). (B and C) Soluble chromatin from RKO/tet-MUC1shRNA (B) and BT-549/tet-MUC1shRNA (C) cells treated with vehicle or DOX for 7 days was precipitated with a control IgG, anti-EZH2 or anti-H3K27me3. The DNA samples were amplified by qPCR with primers for the MICA and MICB promoter regions. The results (mean±SD of three determinations) are expressed as fold enrichment relative to that obtained with the IgG control (assigned a value of 1). (D.) RKO cells treated with 5 μM GSK343 for 6 days were analyzed for MICA and MICB mRNA levels by qRT-PCR (left). The results (mean±SD of four determinations) are expressed as relative mRNA levels compared with that obtained for vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). (E) RKO/tet-MUC1shRNA cells treated with vehicle or DOX for 7 days were analyzed for the indicated DNMT mRNA levels by qRT-PCR (left). The results (mean±SD of four determinations) are expressed as relative mRNA levels compared with that obtained for vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with antibodies against the indicated proteins (right). (F) RKO cells treated with vehicle or 5 μM DEC for 5 days were analyzed for MICA and MICB mRNA levels by qRT-PCR (left). The results (mean±SD of four determinations) are expressed as relative mRNA levels compared with that obtained for vehicle-treated cells (assigned a value of 1). Lysates were immunoblotted with the indicated antibodies (right). (G) RKO cells treated with vehicle or 5 μM DEC for 5 days were analyzed for cell surface MICA and MICB expression by flow cytometry. DEC, decitabine; DNMT, DNA methyltransferase; MICA, MHC class I chain-related polypeptide A; MICB, MHC class I chain-related polypeptide B.

Article Snippet: Immunoprecipitation was performed using a control IgG (3900S, CST) and antibodies against EZH2 (#5246S, CST) and H2K27me3 (#9733S, CST).

Techniques: Expressing, DNA Methylation Assay, Quantitative RT-PCR, Amplification, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: MUC1-C is a master regulator of MICA/B NKG2D ligand and exosome secretion in human cancer cells

doi: 10.1136/jitc-2022-006238

Figure Lengend Snippet: Targeting MUC1-C activates NK cell-mediated killing of cancer cells. (A and B) Control and GO-203-treated RKO (A) and COLO 201 (B) cells were cocultured with human NK cells from two different donors. NK-specific killing was assessed in three different NK cell:tumor cell ratios. The results are expressed as mean±SD of six determinations. (C) Schema of MUC1-C-driven repression of MICA/B expression. MUC1-C activates the PRC2 complex and interacts directly with EZH2 in promoting H3K27 methylation. MUC1-C also induces DNMT expression with increases in DNA methylation. Targeting MUC1-C genetically and with the GO-203 inhibitor suppresses H3K27 and DNA methylation and thereby derepresses MICA/B with increases in cell surface expression. Created by BioRender.com. (D) MUC1-C undergoes processing in the ER for positioning in the cell membrane. In the ER, MUC1-C associates with the ERp5 thiol reductase. GO-203 inhibits that interaction and blocks proteolytic shedding of MICA/B. MUC1-C also interacts with RAB27A in promoting secretion of MICA/B-expressing exosomes, which is inhibited by GO-203 treatment. These results support a model in which targeting MUC1-C promotes NK-mediated recognition and killing of tumor cells. Created by BioRender.com. DNMT, DNA methyltransferase; MICA, MHC class I chain-related polypeptide A; MICB, MHC class I chain-related polypeptide B; NK, natural killer.

Article Snippet: Immunoprecipitation was performed using a control IgG (3900S, CST) and antibodies against EZH2 (#5246S, CST) and H2K27me3 (#9733S, CST).

Techniques: Expressing, Methylation, DNA Methylation Assay

Journal: Cell Death & Disease

Article Title: Differential effects of the LncRNA RNF157-AS1 on epithelial ovarian cancer cells through suppression of DIRAS3- and ULK1-mediated autophagy

doi: 10.1038/s41419-023-05668-5

Figure Lengend Snippet: A Subcellular localization analysis suggested that RNF157-AS1 was localized mainly in the nucleus in EOC cells. B The RPISeq database showed that RNF157-AS1 had a very high interaction predicting score with HMGA1 protein (RF:0.8; SVM:0.88). C RNF157-AS1 expression had a positive correlation with HMGA1 expression ( R = 0.32, P = 2.5*10 −13 ) according to the GEPIA database. D Western blot analysis showed that RNF157-AS1 pulled down a greater amount of protein than did the control. E The RIP assay suggested HMGA1 interacted with RNF157-AS1 in not only RNF157-AS1 overexpression but also control EOC cells. F According to the RPISeq database, EZH2 had the highest interaction predicting score with RNF157-AS1 among the four subunits of the PRC2 complex. G EZH2 had a similar interaction predicting scores between RNF157-AS1 and HOTAIR; H RNF157-AS1 had a positive correlation with HMGA1 ( R = 0.25, P = 5.6*10 −9 ) according to the GEPIA database. I Western blot analysis showed that EZH2 was present only in the RNF157-AS1 (sense) precipitate compared with the control (antisense) precipitate. J The RIP assay suggested EZH2 interacted with RNF157-AS1 in not only RNF157-AS1 overexpressing but also control EOC cells. Data were obtained from at least three independent experiments. *** P < 0.001 ( t -test).

Article Snippet: Anti-EZH2(5246S), anti-LC3B (3868S), and anti-β-actin (4970S) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Expressing, Western Blot, Over Expression

Journal: Cell Death & Disease

Article Title: Differential effects of the LncRNA RNF157-AS1 on epithelial ovarian cancer cells through suppression of DIRAS3- and ULK1-mediated autophagy

doi: 10.1038/s41419-023-05668-5

Figure Lengend Snippet: A Differentially expressed mRNAs after RNF157-AS1 knockdown with hierarchical clustering. B Differentially expressed mRNAs shown on a volcano plot. C and D RNF157-AS1 expression had a negative correlation with DIRAS3 expression ( R = −0.39, P = 1.2*10 −19 ) and ULK1 expression ( R = −0.3, P = 8.4*10 −12 ). E–G The mRNA and protein expression level of DIRAS3 after RNF157-AS1 knockdown or overexpression. Full-length blots are presented in Supplementary Figs. S and S . H–J The mRNA and protein expression level of ULK1 after RNF157-AS1 knockdown or overexpression. Full-length blots are presented in Supplementary Figs. S and S . K and L ChIP-qPCR assay demonstrated that inhibiting RNF157-AS1 suppressed the binding of EZH2 to the promoter of the DIRAS3 genes and that RNF157-AS1 overexpression enhanced the binding of EZH2 to the promoter of the DIRAS3. M and N The ChIP assay demonstrated that diminishing of RNF157-AS1 decreased the binding of HMGA1 to the promoter of ULK1 genes and RNF157-AS1 overexpression enhanced the binding of HMGA1 to the promoter of ULK1. Data were obtained from at least three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001 ( t -test).

Article Snippet: Anti-EZH2(5246S), anti-LC3B (3868S), and anti-β-actin (4970S) antibodies were purchased from Cell Signaling Technology (Boston, MA, USA).

Techniques: Expressing, Over Expression, Binding Assay

Journal: bioRxiv

Article Title: A hyper-quiescent chromatin state formed during aging is reversed by regeneration

doi: 10.1101/2023.02.14.528512

Figure Lengend Snippet: (A) RT-qPCR analysis of Ezh1, Ezh2, Kdm6a and Kdm6b RNA levels in young and old livers. Data are summarized as mean ± S.E.M. (n=8 biological replicates per group). (B) Western blot of H3K27me3, H3, EZH1, EZH2, KDM6A and KDM6B protein levels in young and old livers. (n=3 biological replicates per group). (C) Western blot of H3K27me3, H3, EZH1 and EZH2 in proliferating and quiescent cells over 14 days. Quiescence was induced by contact inhibition (n=2 biological replicates per group). (D) Heatmap showing EZH2 signal at differential peaks from . (E) Genome browser snapshots of EZH2 enrichment over 4 peak regions from (D). (F) Western blot of H3K27me3, H3, EZH1 and EZH2 from different salt fractions of chromatin from in young and old livers. On the right is the Ponceau S-stained membrane from the same experiment.

Article Snippet: EZH2 ChIP was performed using the SimpleChIP plus sonication kit (CST #56383).

Techniques: Quantitative RT-PCR, Western Blot, Inhibition, Staining

Journal: bioRxiv

Article Title: A hyper-quiescent chromatin state formed during aging is reversed by regeneration

doi: 10.1101/2023.02.14.528512

Figure Lengend Snippet: (A) GO terms associated with differential peaks in . Development and differentiation terms are indicated in red. (B) Negative correlation between H3K27me3 and gene expression change in old vs young. (C) H3K27me3 ChIP-seq profiles at indicated loci in young and old livers. (D) BioAnalyzer profiles of fixed chromatin from young and old livers digested with increasing units of MNase. (E) The digestion profile with 2000U of MNase shown as an overlayed trace. (F) Heatmap showing EZH2 signal at all called peaks in young and old livers. (G) ERCC Exfold transcript abundance and overall spike-in strategy. A-D represent the 4 groups of transcripts present in Mix 1 and 2. (H) Observed vs expected plot of Mix1/Mix2 log ratio for one pair of sex-matched young and old animals before resection. The dotted line represents a hypothetical experiment where the observed Mix1/Mix2 ratio is the same as expected. On the right, same data shown as box plots with median values indicated.

Article Snippet: EZH2 ChIP was performed using the SimpleChIP plus sonication kit (CST #56383).

Techniques: Expressing, ChIP-sequencing

Journal: bioRxiv

Article Title: A hyper-quiescent chromatin state formed during aging is reversed by regeneration

doi: 10.1101/2023.02.14.528512

Figure Lengend Snippet: (A) Volcano plot of differentially expressed mRNAs 48 h post-resection in young and old livers, blue dots are significant (p<0.05) mRNAs. Biological process GO terms are indicated for genes downregulated (left) or upregulated (right) in the old. Cell proliferation genes are indicated in red. (B) Same as (A) except samples are 96 h post-resection. (C) qPCR analysis of Mki67, Dnmt1, Ezh2 and Suv39h1 expression relative to Actb across the regeneration time-course. Data are summarized as mean ± S.E.M. (n=3 biological replicates per group per timepoint). * p<0.05, *** p<0.001 and **** p<0.0001 from a two-way ANOVA. (D) Heatmap of cell proliferation gene counts across the regeneration time-course.

Article Snippet: EZH2 ChIP was performed using the SimpleChIP plus sonication kit (CST #56383).

Techniques: Expressing

Journal: bioRxiv

Article Title: A hyper-quiescent chromatin state formed during aging is reversed by regeneration

doi: 10.1101/2023.02.14.528512

Figure Lengend Snippet: (A) Schematic of complete liver regeneration. (B) Western blot of H3K27me3, H3 and β-actin showing replication dilution of H3K27me3 post-regeneration. The “before regeneration” H3K27me3 blot is same as in . (C) Western blot of H3K27me3, H3, EZH1, EZH2 and β-actin post-regeneration. For (B-C) n=3 and 2 replicates per group respectively. The “after regeneration” H3K27me3 blot is same as in (B). (D) Heatmap of H3K27me3 signal at differential peaks from before and after regeneration. (E) Negative correlation of gene expression between old vs young and old regenerated vs old. The gene set corresponds to common genes that were de-repressed in aging and re-repressed post-regeneration. (F) Genome browser shot of overlayed H3K27me3 ChIP signal over chr5 (top) and chr18 (bottom) before and after regeneration. Green area is expanded on the right of each chromosome. (G) PCA plot of H3 subtracted H3K27me3 genome coverage from young, young regenerated, old, and old regenerated livers. (H) Same as (G) except PCA is from RNA-seq data. For (G-H) n=3 replicates per group. (I) Heatmap of liver-enriched gene counts in young, old, and old regenerated livers sorted on the old sample. (J) Heatmap of age-upregulated (from Fig. S9D) gene counts in young, old, and old regenerated livers sorted on the old sample. (K) Overview of H3K27me3 changes in aging and reversal by regeneration.

Article Snippet: EZH2 ChIP was performed using the SimpleChIP plus sonication kit (CST #56383).

Techniques: Western Blot, Expressing, RNA Sequencing Assay