Journal: Frontiers in Chemistry

Article Title: Lethal activity of BRD4 PROTAC degrader QCA570 against bladder cancer cells

doi: 10.3389/fchem.2023.1121724

Figure Lengend Snippet: The data sets collected from public GDSC database.

Article Snippet: Primary antibodies to the following proteins were used: BRD4 (Bethyl Laboratories, A301-985A100), BRD3 (Bethyl Laboratories, A302-368A), BRD2 (Bethyl Laboratories, A302-583A), EZH2 (D2C9) (Cell Signaling Technology Inc, #5246), c-MYC (Abcam, ab32072), caspase-3 (Cell Signaling Technology Inc, #9662) GAPDH (ABclonal, AC033), β-Actin (ABclonal, AC006).

Techniques: Sequencing

Journal: Frontiers in Chemistry

Article Title: Lethal activity of BRD4 PROTAC degrader QCA570 against bladder cancer cells

doi: 10.3389/fchem.2023.1121724

Figure Lengend Snippet: BRD4 target genes are downregulated by QCA570 (A) The EZH2 mRNA expression level of bladder tissues was analyzed in TGCA cohorts. The p -value was examined by the Mann–Whitney U-test. (B) The relative expression level of EZH2 was determined in GEO database (GSE13507). Using the remove Batch Effect function in the limma package to removes batches. The p -value was examined by Welch’s t -test. (C, D) RT-qPCR analysis of c-MYC and EZH2 gene expression in J82 and T24 cells. Values are the means ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed Student’s t-test. (E–G) The protein level of c-MYC and EZH2 were detected by Western blotting. GAPDH was used as loading control.

Article Snippet: Primary antibodies to the following proteins were used: BRD4 (Bethyl Laboratories, A301-985A100), BRD3 (Bethyl Laboratories, A302-368A), BRD2 (Bethyl Laboratories, A302-583A), EZH2 (D2C9) (Cell Signaling Technology Inc, #5246), c-MYC (Abcam, ab32072), caspase-3 (Cell Signaling Technology Inc, #9662) GAPDH (ABclonal, AC033), β-Actin (ABclonal, AC006).

Techniques: Expressing, MANN-WHITNEY, Quantitative RT-PCR, Two Tailed Test, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Discovery of IHMT-337 as a potent irreversible EZH2 inhibitor targeting CDK4 transcription for malignancies

doi: 10.1038/s41392-022-01240-3

Figure Lengend Snippet: Characterization of IHMT-337 as a highly selective EZH2 inhibitor. a Chemical structure of IHMT-337. b EZH2 signaling studies: Target effects of IHMT-337 on EZH2 signaling in Pfeiffer and Karpas422 cell lines. EPZ6438 (the FDA-approved EZH2 inhibitor) was set as control. c Proliferation studies: Effects of 6-day IHMT-337 treatment of Pfeiffer,Karpas422 and SU-DHL6 cell lines. EPZ6438 was set as control. d The GI50 values (the concentrations that cause 50% growth inhibition) of IHMT-337 and EZP6438 to DLBCL cell lines were shown. e Biochemical assays: the effects of IHMT-337 on EZH2 methyltransferase activity on PRC2/EZH2 complex. f Methyltransferase selectivity profiling of IHMT-337 generated from the Hotpot approach. Data shown were representative of at least 2 independent experiments

Article Snippet: The EZH2 (D2C9) Rabbit mAb (#5246), SUZ12 (D39F6) XP® Rabbit mAb (#3737), PARP (46D11) Rabbit mAb (#9532), Ubiquitin (P4D1) Mouse mAb (#3936), CDK4 (D9G3E) Rabbit mAb (#12790), Phospho-Rb (Ser807/811) (D20B12) Rabbit mAb (#8516), Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (#9733), Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (#9751), Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (#13969) and Tri-Methyl-Histone H3 (Lys79) (E8B3M) Rabbit mAb (#74073) were obtained from Cell Signaling Technology.

Techniques: Inhibition, Activity Assay, Generated

Journal: Signal Transduction and Targeted Therapy

Article Title: Discovery of IHMT-337 as a potent irreversible EZH2 inhibitor targeting CDK4 transcription for malignancies

doi: 10.1038/s41392-022-01240-3

Figure Lengend Snippet: IHMT-337 covalently binds to EZH2 at Cys663 residue in SET domain. a The CETSA assay: The effect of IHMT-337 on the stability of the EZH2 protein in a temperature-dependent manner was investigated using WSU-DLCL2 cell lysate. b The CETSA assay: The effect of IHMT-337 on the stability of the EZH2 protein in a dose-dependent manner was investigated using WSU-DLCL2 cell lysate. c Washout assay: The effect of washout assay on signal pathway inhibition post-drug washout at different time points after using IHMT-337 and IHMT-338 treatment 72 h on WSU-DLCL2 cell line. d Target-engagement assay: Using Biotin-IHMT-337 and IHMT-337 to investigate the binding of IHMT-337 to EZH2 in Pfeiffer cells. e Predicted mode of binding of IHMT-337 to EZH2 based upon molecular modeling (PDB ID 5IJ7, chain B). f Using the HEK293T EZH2-KO cell line and plasmids with different mutations, investigation of the contribution of three cysteines in the SET domain to the direct binding of EZH2 and IHMT-337, the wt EZH2 was set as control. g The level of H3K27me3 was quantified and graphed. Shown are the representative results of three independent experiments

Article Snippet: The EZH2 (D2C9) Rabbit mAb (#5246), SUZ12 (D39F6) XP® Rabbit mAb (#3737), PARP (46D11) Rabbit mAb (#9532), Ubiquitin (P4D1) Mouse mAb (#3936), CDK4 (D9G3E) Rabbit mAb (#12790), Phospho-Rb (Ser807/811) (D20B12) Rabbit mAb (#8516), Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (#9733), Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (#9751), Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (#13969) and Tri-Methyl-Histone H3 (Lys79) (E8B3M) Rabbit mAb (#74073) were obtained from Cell Signaling Technology.

Techniques: Inhibition, Binding Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Discovery of IHMT-337 as a potent irreversible EZH2 inhibitor targeting CDK4 transcription for malignancies

doi: 10.1038/s41392-022-01240-3

Figure Lengend Snippet: IHMT-337 degrades EZH2 via CHIP-mediated ubiquitination pathway. a Effects of 24 h IHMT-337 treatment on EZH2 protein levels in both Pfeiffer and MDA-MB-231 cells. b (Left panel) Pfeiffer and MDA-MB-231 cells were treated with CHX (40 μg/ml) with or without IHMT-337 treatment (10 μM) at the indicated time points. EZH2 and GAPDH protein levels were detected by western blotting. (Right panel) The half-life of EZH2 protein was quantified and graphed. Shown are the representative results of three independent experiments. c Pfeiffer and MDA-MB-231 cells were treated with IHMT-337 and with or without the proteasome inhibitor, MG132 (5 μM) at the indicated time points, EZH2 and GAPDH protein levels were detected by western blotting. d Pfeiffer and MDA-MB-231 cells were treated with IHMT-337 for 24 h at 0, 2.5, 5, and 10 μM. IP was performed with antibodies against EZH2, ubiquitin, EZH2, and GAPDH protein levels were detected by western blotting. e Cell lysates from Pfeiffer or MDA-MB-231 cells were treated with Biotin-IHMT-337 for 4 h at 0, 1 μM. IP was performed with Streptavidin bead through streptavidin-biotin interaction, and immunoblotting was performed with antibodies against EZH2 and CHIP

Article Snippet: The EZH2 (D2C9) Rabbit mAb (#5246), SUZ12 (D39F6) XP® Rabbit mAb (#3737), PARP (46D11) Rabbit mAb (#9532), Ubiquitin (P4D1) Mouse mAb (#3936), CDK4 (D9G3E) Rabbit mAb (#12790), Phospho-Rb (Ser807/811) (D20B12) Rabbit mAb (#8516), Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (#9733), Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (#9751), Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (#13969) and Tri-Methyl-Histone H3 (Lys79) (E8B3M) Rabbit mAb (#74073) were obtained from Cell Signaling Technology.

Techniques: Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Discovery of IHMT-337 as a potent irreversible EZH2 inhibitor targeting CDK4 transcription for malignancies

doi: 10.1038/s41392-022-01240-3

Figure Lengend Snippet: IHMT-337 inhibits breast cancer cell proliferation by degrading EZH2, a CDK4 transcription factor. a Proliferation studies: Effects of 6-day IHMT-337 treatment on proliferation of TNBC cell lines. EPZ6438 was set as control. b Proliferation studies: Effects of EZH2 knockdown on proliferation of MDA-MB-231 cells. c Cell cycle studies: Effects of IHMT-337 on cell cycle in MDA-MB-231 cell. EPZ6438 was set as control. d The CUT&TAG approach was used on HEK293T and HEK293T EZH2-KO cell lines to determine the sites of EZH2 binding to DNA. e Signaling studies: The inhibitory Effects of 72 h IHMT-337 treatment on cell cycle signaling in MDA-MB-231 cells. EPZ6438 was set as control. f Effects of 72 h IHMT-337 treatment of MDA-MB-231 cells on CDK4 transcriptional level. g Protein levels of EZH2 in HEK239T WT, HEK293T EZH2-KO, and HEK293T SUZ12 KO cells. h Transcriptional level of CDK4 in HEK239T WT, HEK293T EZH2-KO, and HEK293T SUZ12 KO cells

Article Snippet: The EZH2 (D2C9) Rabbit mAb (#5246), SUZ12 (D39F6) XP® Rabbit mAb (#3737), PARP (46D11) Rabbit mAb (#9532), Ubiquitin (P4D1) Mouse mAb (#3936), CDK4 (D9G3E) Rabbit mAb (#12790), Phospho-Rb (Ser807/811) (D20B12) Rabbit mAb (#8516), Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (#9733), Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (#9751), Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (#13969) and Tri-Methyl-Histone H3 (Lys79) (E8B3M) Rabbit mAb (#74073) were obtained from Cell Signaling Technology.

Techniques: Binding Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Discovery of IHMT-337 as a potent irreversible EZH2 inhibitor targeting CDK4 transcription for malignancies

doi: 10.1038/s41392-022-01240-3

Figure Lengend Snippet: IHMT-337 inhibits cell proliferation in different preclinical models in vitro and in vivo. a Body weight change in mice for each twice-daily dosing group of IHMT-337 and EPZ6438. Initial body weight was set as 100%. Comparison of the final tumor weight in each group of 22-day treatment period. b Relative tumor size measurements of Pfeiffer xenograft mice after IHMT-337 and EPZ6438 treatment. c Effects of 22 days IHMT-337 treatment on growth of Pfeiffer xenograft tumor model were determined. EPZ6438 was set as control. d Effects of 72 h IHMT-337 treatment on TNBC PDO models. e The inhibitory effects of IHMT-337 on protein levels of EZH2 and CDK4 in TNBC PDOs were determined by confocal assays. f The inhibitory effects of IHMT-337 on proliferation of TNBC PDOs were determined. EPZ6438 was set as control. g Transcriptional level of CDK4 in TNBC PDOs with or without IHMT-337 treatment were determined by Q-PCR. h IHMT-337 affects cell cycle progression through targeting transcriptional regulating of CDK4

Article Snippet: The EZH2 (D2C9) Rabbit mAb (#5246), SUZ12 (D39F6) XP® Rabbit mAb (#3737), PARP (46D11) Rabbit mAb (#9532), Ubiquitin (P4D1) Mouse mAb (#3936), CDK4 (D9G3E) Rabbit mAb (#12790), Phospho-Rb (Ser807/811) (D20B12) Rabbit mAb (#8516), Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit mAb (#9733), Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb (#9751), Tri-Methyl-Histone H3 (Lys9) (D4W1U) Rabbit mAb (#13969) and Tri-Methyl-Histone H3 (Lys79) (E8B3M) Rabbit mAb (#74073) were obtained from Cell Signaling Technology.

Techniques: In Vitro, In Vivo

Journal: Nature Communications

Article Title: PCGF1-PRC1 links chromatin repression with DNA replication during hematopoietic cell lineage commitment

doi: 10.1038/s41467-022-34856-8

Figure Lengend Snippet: a Target binding of KDM2B, BCOR, RING1B, H2AK119ub1, JARID2, SUZ12, H3K27me3 and PHC2 in control (Ctrl) and Pcgf1 -KO (KO) IdHPCs. A heatmap of ChIP-seq signals across TSS( ± 10 kb) of C1, C2, C3, and C4 genes in control and Pcgf1 -KO is shown. Local levels of RING1B and H2AK119ub1 were also tested in Ring1a/b -dKO (R1ABdKO) IdHPCs as controls. H3K27me3 ChIP-seq were calibrated by spike-in chromatin. Representative data of biological duplicates are shown except for RING1B ChIP-seq in Ring1a/b- dKO IdHPCs, which was obtained from a single experiment. b Box plot views for ChIP-seq results across TSS ( ± 5 kb) for RING1B, H2AK119ub1, SUZ12 and H3K27me3 in each cluster in control, Pcgf1 -KO and (in the case of H2AK119ub1) Ring1a/b- dKO IdHPCs. Data in graphs represent means for two biologically independent experiments. The center circle indicates a median value and the boxes indicate 25th to 75th percentile. Each dot represents individual genes. The numbers beneath the graph are p -values between the control and Pcgf1-KO calculated with the Wilcoxon signed rank test. CPM: Counts Per Million. c ChIP-qPCR analyses for local binding of RING1B, H2AK119ub1, SUZ12 (PRC2), EZH2 (PRC2), H3K27me3, PHC2 (cPRC1) and BMI1 (cPRC1) at selected C1 and C2 genes in the control and Pcgf1 -KO IdHPCs. Data represent mean ± SD of three independent experiments, except for ChIP for EZH2 which is derived from two independent analysis. The numbers on the graph are p -values between the control and Pcgf1 -KO calculated with the Student’s two-sided t test.

Article Snippet: anti EZH2(D2C9) (Cell Signaling, #5246) , ChIP (1 : 50), IB (1 : 1000).

Techniques: Binding Assay, ChIP-sequencing, Derivative Assay

Journal: Nature Communications

Article Title: PCGF1-PRC1 links chromatin repression with DNA replication during hematopoietic cell lineage commitment

doi: 10.1038/s41467-022-34856-8

Figure Lengend Snippet:

Article Snippet: anti EZH2(D2C9) (Cell Signaling, #5246) , ChIP (1 : 50), IB (1 : 1000).

Techniques:

Journal: bioRxiv

Article Title: lncRNA H19/Let7b/EZH2 axis regulates somatic cell senescence

doi: 10.1101/2022.07.07.499142

Figure Lengend Snippet: A Let-7b binding sites in the 3’UTR of EZH2 mRNA as predicted by TargetScan 6.0. b EZH2 mRNA expression was determined by RT-qPCR in the early passage, late passage, and late passage human cardiac fibroblasts (HCF) treated with rapamycin (Mean±SEM, *p < 0.05 by two-tailed unpaired Student’s t -test, n = 3). C EZH2 protein expression was analyzed by western blot in the early, late, and late passage human cardiac fibroblasts (HCF) treated with rapamycin. α/β tubulin serves as the loading control. D Early passage cells grown with or without rapamycin-containing media were treated with an EZH2 inhibitor, GSK343, and growth was assessed by cumulative population doublings (cPD). E Early passage cells were transfected with control or let-7b mimic, and EZH2 mRNA expression was determined by RT-qPCR (Mean±SEM, **p < 0.01 by two-tailed unpaired Student’s t -test, n = 3). F EZH2 protein expression was analyzed in early passage cells transfected with control or let7b overexpression vector, and EZH2 mRNA expression was determined by RT-qPCR. G Early passage HCF cells were transfected with siRNA targeting H19 (siH19) and negative control (siNeg), and EZH2 mRNA were determined 7 days after transfection (Mean±SEM, ***p < 0.001 by two-tailed unpaired Student’s t -test, n = 3). H EZH2 protein levels were analyzed in siH19 and siNeg transfected cells. I Representative tracks from H3K27me3 CUT&Tag-sequencing for p21 Cip1/Waf1 (CDKN1A) gene. J Representative tracks from H3K27me3 CUT&Tag-sequencing for p16 INK4A (CDKN2A) gene. Tracks show H3K27me3 marks at the p16 INK4A gene for early and late passage HCF cells in teal and yellow, respectively. The Refseq gene track is displayed in grey. Source data are provided as a Source Data file.

Article Snippet: Antibodies used were CTCF (D31H2) XP(R) Rabbit monoclonal antibody (Cat. 3418S, Cell Signaling Tech; 1:1000 dilution), EZH2 (D2C9) XP(R) Rabbit monoclonal antibody (Cat. 5246S, Cell Signaling Tech; 1:1000 dilution), p21Cip1/Waf1 Waf1/Cip1 (12D1) Rabbit monoclonal antibody (Cat.2947S, Cell Signaling Tech; 1:1000 dilution), p16 INK4A (JC8) mouse monoclonal antibody (Cat.sc-56330, Santa Cruz; 1:1000 dilution), p53 monoclonal antibody (Cat. 39554, ActiveMotif; 1:1000 dilution), α/β tubulin Rabbit antibody (Cat. 2148S, Cell Signaling Tech; 1:1000 dilution), GAPDH (D16H11) XP(R) Rabbit monoclonal antibody (Cat. 5174S, Cell Signaling Tech; 1:1000 dilution)

Techniques: Binding Assay, Expressing, Quantitative RT-PCR, Two Tailed Test, Western Blot, Transfection, Over Expression, Plasmid Preparation, Negative Control, Sequencing

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Schematic representation of steps in FLASH-seq (formaldehyde and UV cross-linking, ligation, a nd s equencing of h ybrids) with EZH2 immunoprecipitation using lysates from UV crosslinked endothelial cells. Dynamic EZH2-RNA complex formation occurs as represented. Following RNA ligation and chimera formation between interacting RNAs, sequencing is performed. Further analysis of single and hybrid reads bound by EZH2, reveals interacting RNA molecules. b. Distribution of annotated reads over genome, with gene classification (biotype), from statistically filtered EZH2-FLASH data with two biological replicates in HUVECs and MEG3-lncRNA (yellow wedge) as the candidate. c. I and ii Enriched motifs with sequences in MEG3 mRNA of EZH2-FLASH that uniquely overlap exons; the logos were drawn using the top 4-8nucleotides K-mers for each experimental replicate ( top and middle ) and z-score for each. Motif analysis was performed using the MEME suite (Bailey et al., 2009) iii : Enriched motif within the fragments of MEG3:MEG3 hybrids d. Total RNA-RNA interactions associated with MEG3 at chr14:101292445-101327360, MEG3 id = NR_002766.2 ) and distribution of all MEG3 interactions among various classes of RNAs as captured by EZH2-FLASH. e. Intermolecular MEG3-RNA interactions found in chimeras captured by EZH2-FLASH. Chimera counts were mapped for all genomic features of annotated hybrids and the ones of MEG3 were plotted in the circos plot with position along the MEG3 genomic sequence. The main MEG3 hybrid is MEG3 and are represented by the number of interactions in red. The feature as a line: Red circle shows the position in the MEG3 gene in kilobases with * 50-55kb falling within exon3; Blue circle is a visual representation of MEG3 exons. Regions overlapping exons are represented in solid blue. Purple broad circle shows the nucleotides. The nucleotides at each position are: A : dark blue, C : light blue, T : light red, G : dark red. The details on the feature: The inner part of the white circle shows MEG3:MEG3 hybrids; Arcs connecting the centre of each hybrid fragment are shown in red, and the regions spanned by the hybrid fragments are shown in light green.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Ligation, Immunoprecipitation, Sequencing

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Distribution of annotated single hits over MEG3 gene, with statistically filtered EZH2-FLASH reads from two biological replicates in HUVECs. b) The occupancy of EZH2 hits over MEG3 features. Total reads per feature are given with exons being mostly occupies vs introns. c) Proportion of overlapping features over MEG3. The occupancy of EZH2 over each MEG3 exon is shown for two constitutively expressed transcripts. For both given transcripts there is high occupancy of exon 3. d) RNA immunoprecipitation (RIP) for EZH2 and H3K27me3 (repressive chromatin) followed by qPCR analysis. RIP-purified RNA from UV crosslinked HUVECs was used to prepare cDNA for qPCR analysis with primers against MEG3 (exon 3 region). Primers against U1snRNA gene serves as a negative control. Side diagram of EHZ2-MEG3 interacting region is charted as per FLASH hits and sequence. e) Distribution of EZH2 hybrids hits over MEG3 gene. Intermolecular MEG3-RNA interactions found in chimeras are captured by EZH2-FLASH-seq. Hits represent MEG3:MEG3 hybrids (black). IgG hybrids are plotted but are <1. f) Total MEG3:MEG3 hybrid count against predicted free energy of hybridization (dG) for MEG3 interactions ( red lncRNA:MEG3, blue mRNA:MEG3, green MEG3:antisense, purple snoRNA:MEG3) with free hybridization energy cutoff at dG<-10 kcal mol -1 , as captured by EZH2-FLASH-seq ( i ) vs. IgG control ( ii ) .

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Immunoprecipitation, Purification, Negative Control, Sequencing, Hybridization

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Overview of the design of probes against MEG3 gene that were divided in probe Set1 and Set 2. The biotynilated probes were of 20 nucleotides and were spaced out 200 nucleotides apart down the gene length. b) Validation of MEG3 probes specifically binding MEG3 gene, by ChIRP-qPCR in HUVECs. Pull down with probe set 1 or set 2 retrieved 100% and 40% RNA, respectively. GAPDH primers were used as control and MEG3-associated samples did not amplify. c) Computational analysis pipeline for ChIRP-seq outlining data processing. The peak coverage was within the 100bp window. d) MEG3-ChIRP peaks associated with EZH2 gene as precipitated using both sets of probes (set 1 and 2). e) Enrichment of MEG3 signal by ChIRP-qpcr versus negative control (Background) at named promoter regions. MEG3 binding to genomic loci as validate by ChIRP-qPCR in HUVECs. Pull downs were performed with joined Odd and Even probes. Value 1 is a background level, defined by enrichment to LacZ negative probes in ChIRP. Control primers were designed for positive ChIRP peaks and used as a positive control and for regions deprived of MEG3-ChIRP reads as a negative control .

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Binding Assay, Negative Control, Positive Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Overview of the critical steps to obtain MEG3-bound genomic loci and intersections with EZH2 and H3K27me3 signals (obtained from GEO databases for HUVECs). In addition, enhancer regions were mapped within the genomic tracks. The intersection between GEO EZH2 ChIP data, GEO H3K27me3 ChIP data and statistically filtered MEG3-ChIRP data from two biological replicates was performed. The number of genes and degree of overlap is obtained between MEG3 and PRC2-dependent genes. The p-values are a result of hypergeometric test. b. Distribution of MEG3 peaks overlapping EZH2-ChIP peaks or H3K27me3-peaks with intersecting reads in relation to (i) gene regions and (ii) gene-type. c. Maximum peak score of ChIP signal for EZH2 and H3K27me3 intersecting the top enriched MEG3 peaks associated with nearest genes. Highest EZH2 peak score is over ITGA4, whereas H3K27me3 was detected in ITGA4, ITGA7, ITGA8 and ITGA9, members of ITGA family. d. Normalized reads from RNA-seq de novo analysis of GEO: GSE71164 dataset on Hg38, and expression of ITGA4 gene between Scr and siEZH2 depleted HUVECs, showing that ITGA4 is targeted by EZH2. Dataset in d and e is compared using Student’s t-test. e. ITGA4 expression from microarray analysis in C2C12 cells depleted of MEG3 (10nM, LNA GapMer) as per GEO dataset: GSE73524. The data shows that ITGA4 is a direct target of MEG3. f. (i) Total number of representable peaks (mRNA, antisense and lncRNA genes) from ChIP-seq analysis of Scr vs. MEG3 KD HUVECs. (ii ) Depletion of MEG3 gene in HUVECs (10nM LNA gapmers) was achieved with relative expression showing ∼70% reduction compared with Scr control. g. (i) Heat map showing distribution of reads and EZH2 densities at all unique RefSeq genes within TSSs ± 3 kb, sorted by EZH2 occupancy, in Control vs. MEG3 deficient (10nM) HUVECs. (ii) Overlap of ChIP-results between MEG3 and EZH2-dependent genes, with overlapped genes belonging to the biological pathway regulating cell adhesion. The common targets had lost or reduced EZH2 ChIP-signal.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing Assay, Expressing, Microarray, ChIP-sequencing

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a ) RNA-seq dataset from HUVEC cells depleted in EZH2 (GSE71164) was de novo analysed and mapped onto Hg38 with reads given in the table. The principal component analysis (PCA) was used to describe the variance between two groups (ctr vs . siEZH2); depletion of EZH2 gene is represented between samples (n=3) with reads per sample, in the bottom table. b ) Heatmap of selected genes directly regulated by EZH2 and involved in angiogenesis and cell adhesion processes.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing Assay

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a) Computational analysis pipeline used to obtain orthologous peaks in human and intersect regions and genes enriched in repressive chromatin (H3K27me3) from ChIP-seq public dataset GSE114283. Up- and down-regulated genes were obtained associated with the peak region within 2000bp, and relevant function and biological pathway were associated using GREAT and DAVID analysis b) Overlap of the GEO datasets from a (Microarray GSE73524 ) and b (RNA-seq GSE71164 ) and the GSE114283 ChIP-seq reads of H3K27me 3 distribution in mouse MN cells depleted of MEG3 vs. control. ChIP extracted peaks unique to Ctrl vs. MEG3 KD were obtained, and associated mouse gene list composed based on reduction in H3K27me 3 signal. Using gene orthologous analysis in gProfiler we obtained human orthologous targets that was used for data intersection. c) Maximum peak scores of the overlapping signal over ITGA4 promoter, obtained by intersection of EZH2 ChIP signal with MEG3-ChIRP signal at this region. Upon depletion of MEG3 the EZH2 signal is significantly reduced whereby no overlap with MEG3 ChIRP signal is seen. d) Relative expression of ITGA4 in HUVEC measuring the levels of ITGA4 following addition of siRNA (50nM).

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: ChIP-sequencing, Microarray, RNA Sequencing Assay, Expressing

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet:

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques:

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Venn diagram showing the intersection between statistically filtered FLASH data from two biological replicates of our MEG3-ChIRP-seq-data (green), de novo hg38 analysed GEO RNA-seq data from siEZH2 deficient HUVECs (GSE71164, blue), and EZH2 ChIP-seq following MEG3 KD (yellow) and FLASH-seq transcriptome following EZH2 IP (pink). b. Correlation between gene expression levels and FLASH signal. Gray, expressed RefSeq genes with reproducible FLASH signal consistently detected in RNA-seq. Blue, genes with the highest RNA-seq signals and no reproducible FLASH signal belonging to integrin cell surface interaction pathway. Red , expressed ITGA4 gene, and green, ITGB1 gene, without reproducible FLASH signals. Data are from two biological replicates of each EZH2 FLASH sample and three biological replicates of EZH2 RNA-seq samples (Scr vs. siEZH2, GSE71164). c. Genomic tracks showing ChIRP-seq signal (MEG3 Odd, Even and LacZ) in HUVECs over ITGA4 gene only. The MEG3 binding site is located upstream of the ITGA4 gene in the promoter region, and it overlaps with the H3K27me3 signal and EZH2; as well as downstream within the ITGA4 gene body, where it overlaps with within the EZH2 signal in the intronic region of the gene. d. MEG3-ChIRP followed by qPCR, analysis of MEG3 binding region on ITGA4 in HUVECs. The crosslinked cell lysates were incubated with combined biotinylated probes against MEG3 lncRNA and the binding complexes recovered by magnetic streptavidin-conjugated beads. The qPCR was performed to detect the enrichment of specific region that associated with MEG3, peaks were related to input control and compared vs. the non-biotynilated control. e. ChIP-QPCR enrichment for EZH2 and H3K27me3 over ITGA4 promoter region in HUVECs depleted of MEG3 vs. Control.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: RNA Sequencing Assay, ChIP-sequencing, Expressing, Binding Assay, Incubation

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. ChIP signal enrichment vs . 1% input for EZH2 and H3K27me3 mark over ITGA4 promoter regions in HUVECs treated with A-395 (5µM, 24h) inhibitor of PRC2 vs. Control (DMSO). The expression was measured using two sets of primers against the same promoter region of ITGA4. Representative graphs are average of three qPCR datasets ± SEM. b. ITGA4 expression in the presence of A-395 vs . DMSO control, N=6 independent experiments compared using t -test. c. Measuring the expression levels of ITGA4 upon depletion of MEG3 using LNA GapmeRs (10nM, 48h), data is mean of N=5 independent experiments (biological replicates). d. Representative image of immunofluorescence staining for ITGA4 protein levels in ECs treated with A-395 vs . DMSO, or upon MEG3 depletion like in b . e. Intra-cellular localisation of MEG3 (chromatin associated lncRNA) between different cellular compartments in HUVECs treated with A-395 vs. DMSO, whereby the distribution of MEG3 has shifted upon PRC2 inhibition with A-395; from the nucleus (where it was highly chromatin bound) into the cytoplasm. Representative bars were compared by t-test and on-way Anova. f. MEG3-ChIRP followed by qPCR, N =3, analysis of MEG3 binding over ITGA4 promoter region in HUVECs treated with A-395 (5µM, 24h) vs. DMSO. MEG3-ChIRP HUVEC lysates treated with A-395 resulted in reduced engagement of MEG3 with ITGA4 site compared with either DMSO control or ChIRP with non-biotinylated probes. The non-biotin probes served as a negative control, and we detected the background level <1.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Expressing, Immunofluorescence, Staining, Inhibition, Binding Assay, Negative Control

Journal: bioRxiv

Article Title: Histone H3K27 methyltransferase EZH2 interacts with MEG3-lncRNA to directly regulate integrin signaling and endothelial cell function

doi: 10.1101/2022.05.20.492787

Figure Lengend Snippet: a. Measure of cell migratory capacity using ECIS functional analysis in ECs treated with control or A-395 (5µM, 24h) inhibitor. Experiments were performed in duplicates (technical replicates) and four experiments were run for migration assay and six for adhesion (biological replicates). The data showing ECIS trace (left hand side) is mean ±SD as calculated by the ECIS. The graph on the right is mean±SEM with N =6, data was compared using ordinary one-way ANOVA with Dunnett’s multiple comparisons tests. b. Adhesion to Fibronectin, FN (20µg/ml) was used to coat the culture plates and assess adhesion of endothelial cells within 3h of ECIS assay, following cell pre-treatment with A-395, 24h. The difference in resistance change was calculated over 3h. c. Subcutaneous Matrigel plug injection (200µl) into mice ( N =5) treated with DMSO (control, left flange) and A-395 (1mg/ml, right flange) was done for 2 weeks. Matrigel plugs were collected and processed for histology. Staining for H3K27me3 was done, displaying nuclear positivity with strong intensity in control (<0.02% DMSO in water) and the A-395 treatment decreased total H3K27me3 staining, as compared by t-test. d. Staining for arterioles was performed to assess vessel growth as angiogenesis and data was compared using Student’s t-test. The data shows increased area of staining for Isolectin B4 (Iso-B4) dye in A-395 vs. DMSO treated Matrigel plugs with increased neovascularization, P<0.05. e. A-395 has increased the percentage of vessels positive for ITGA4 (red) within the Isolectin B4 positive cells, compared with the DMSO using t -test. f. Graphical abstract. 1 Maternally Expressed Gene–MEG3 is highly expressed with hypoxia and bound to EZH2 in endothelial cells (EC) affected by ischaemic insult. 2 Such MEG3:EZH2 complex assembles onto the target genes to 3 direct the EZH2 activity to “write” H3K27me3 trimethylation repressive mark and block expression of target gene i.e. integrin alpha 4 (ITGA4) and its ability to dimerise with integrin beta 1 (ITGB1), leading to 4 reduced EC function as measured by adhesion and migration. Hence 5 targeted disruptions of MEG3:EZH2 interaction, or inhibition of EZH2 activity could increase EC function under ischaemia.

Article Snippet: Following sonication as described, samples were immunoprecipitated using EZH2 (D2C9) XP(R) Rabbit mAb, (5246S Cell signalling technology), Tri-Methyl-Histone H3 (H3K27me3) (C36B11) Rabbit mAb (9733S, CST) antibodies or IgG control (Normal Rabbit IgG, 2729S, CST) and captured on beads using Protein G Dyneabeads (10003D, Life Technologies).

Techniques: Functional Assay, Migration, Injection, Staining, Activity Assay, Blocking Assay, Expressing, Inhibition

Journal: Nature Communications

Article Title: The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer

doi: 10.1038/s41467-021-26901-9

Figure Lengend Snippet: A Sequenced NEPC clinical cohorts. In the VPC cohort ( n = 75), rising levels of H19 expression are seen across increasing Gleason grades (Gleason grading ≦ 6 = AD Low and Gleason grading ≧ 8 = AD High), including NHT treated samples and peaks in NEPC. Significant upregulation of H19 is observed in mixed Gleason grading (MX-G) adenocarcinoma vs. NEPC in the WCM1 cohort ( n = 37) and CRPC vs. NEPC samples of WCM2 ( n = 49) and WCDT ( n = 45) cohorts. Non-significant (ns) yet the elevated expression of H19 is observed in benign (BE) vs. dNEPC of the BCCA cohort ( n = 15), yet possibly due to high tumor cellularity in matched BE samples. B Microarray NEPC clinical cohorts. Similarly, in the JHMI ( n = 33) and GRID ( n = 526) cohorts, rising levels of H19 from AD High/AD MX-G to mixed AD and small cell pathology (MX-P) to NEPC are observed. Box and Whisker plots display lower quartile, upper quartile, and median bounds of cohort expression at the box’s minima, maxima, and centerlines, respectively. Whisker lines display lower (bottom) and upper (top) extreme value ranges. Single data points represent outliers in a cohort. p Values were calculated by an unpaired two-sided Student’s t test. Significance was represented by * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001 unless specifically noted. C In WCM1, H19 shows a significant positive correlation with CHGA/B, SYP, SOX2, and EZH2 and shows a significant negative correlation with AR expression. D Again in WCM1, H19 shows a significant positive and negative correlation to known NEPC and AR gene signatures, respectively. Correlation coefficients ( R ) and p values ( p ) were calculated using a Pearson correlation statistical test. The shaded area represents confidence intervals at 95%. E Unsupervised hierarchical clustering of 38 known genes/lncRNAs in our NEPC ( n = 50) and AD MX-G ( n = 86) samples merged across all cohorts show a clear stratification of these two phenotypes. Select genes denoted by arrows have been shown in our correlation analysis from panel C and Supplementary Fig. .

Article Snippet: Antibodies used: SOX2 (sc-365823, Santa Cruz Biotechnology, 1:400 dilution), H3K27me3 (9733S, Cell Signaling Technology, 1:1000 dilution), EZH2 ((D2C9) XP Rabbit mAb (5246, Cell Signaling Technology, 1:1000 dilution), NSE (sc-21738, Santa Cruz Biotechnology, 1:500 dilution), Synaptophysin (sc-365488, Santa Cruz Biotechnology, 1:500 dilution), CHGA (60893S, Cell Signaling Technology, 1:1000 dilution), BRN2 ((D2C1L) Rabbit mAb 12137, Cell Signaling Technology, 1:1000 dilution), H3 ((D1H2) XP ® Rabbit mAb 4499, Cell Signaling Technology, 1:1000 dilution), Androgen receptor (5153S, Cell Signaling Technology, 1:1000 dilution), H3K4me3 (ab8580, abcam, 1:1000 dilution), P53 (2527 S, Cell Signaling Technology, 1:1000 dilution), Rb (9313S, Cell Signaling Technology, 1:1000 dilution), CK8 (sc–57004, Santa Cruz Biotechnology, 1:300 dilution), PSA (sc-7316, Santa Cruz Biotechnology, 1:250 dilution), HRP conjugated anti-β-actin (Cat. no. A3854, Sigma, 1:10,000 dilution).

Techniques: Expressing, Microarray, Whisker Assay

Journal: Nature Communications

Article Title: The long noncoding RNA H19 regulates tumor plasticity in neuroendocrine prostate cancer

doi: 10.1038/s41467-021-26901-9

Figure Lengend Snippet: A Nuclear localization of H19 in NCI-H660. y -Axis represents the percent abundance of RNA. Nuclear U1 RNA was used as a control. B WB of NE associated genes (SOX2, CHGA, BRN2, and EZH2), H3K27me3, and H3K4me3 in various AdPC and NEPC cell lines and organoids. C WB of H3K27me3, H3K4me3 in CRPC cell line V16D CRPC and AdPC cell line LNCaP after transient overexpression of H19 . The bar graph shows the relative H19 RNA levels in both the cell lines upon H19 overexpression. 18S was used as an endogenous control. D WB analysis of the levels of EZH2 (Actin as control) and histone H3K27me3 level (Histone H3 as control) in Control (Lv-Scr) and H19 knockdown (Lv-shH19) OWCM-155 NEPC organoids. Numerical values shown under the blot are calculated relative to the control samples. E Relative enrichment of H19 binding to PRC2 complex members EZH2, SUZ12 in LASCPC-01, LNCaP, and V16D CRPC cells with transient overexpression of control (EV) and H19 (H19). F WB analysis of LNCaP cells stably expressing doxycycline (DOX) inducible H19 FL (full-length H19 ) or H19 DEL (5′ deleted H19 fragment) with or without DOX treatment (200 ng/mL, 48 h). Actin was used as a control. Data are mean ± SD ( A , C ), or mean ± SEM ( E ); n = 3 ( A , C , E ) biologically independent replicates. p Values were calculated by unpaired two-tailed Student’s t test.

Article Snippet: Antibodies used: SOX2 (sc-365823, Santa Cruz Biotechnology, 1:400 dilution), H3K27me3 (9733S, Cell Signaling Technology, 1:1000 dilution), EZH2 ((D2C9) XP Rabbit mAb (5246, Cell Signaling Technology, 1:1000 dilution), NSE (sc-21738, Santa Cruz Biotechnology, 1:500 dilution), Synaptophysin (sc-365488, Santa Cruz Biotechnology, 1:500 dilution), CHGA (60893S, Cell Signaling Technology, 1:1000 dilution), BRN2 ((D2C1L) Rabbit mAb 12137, Cell Signaling Technology, 1:1000 dilution), H3 ((D1H2) XP ® Rabbit mAb 4499, Cell Signaling Technology, 1:1000 dilution), Androgen receptor (5153S, Cell Signaling Technology, 1:1000 dilution), H3K4me3 (ab8580, abcam, 1:1000 dilution), P53 (2527 S, Cell Signaling Technology, 1:1000 dilution), Rb (9313S, Cell Signaling Technology, 1:1000 dilution), CK8 (sc–57004, Santa Cruz Biotechnology, 1:300 dilution), PSA (sc-7316, Santa Cruz Biotechnology, 1:250 dilution), HRP conjugated anti-β-actin (Cat. no. A3854, Sigma, 1:10,000 dilution).

Techniques: Over Expression, Binding Assay, Stable Transfection, Expressing, Two Tailed Test

Journal: Molecular cancer therapeutics

Article Title: CDK9 Blockade Exploits Context-Dependent Transcriptional Changes to Improve Activity and Limit Toxicity of Mithramycin for Ewing Sarcoma

doi: 10.1158/1535-7163.MCT-19-0775

Figure Lengend Snippet: The combination of mithramycin and PHA-767491 reverses the activity of EWS-FLI1. A and B, 100 nM mithramycin for 18 hours blocks the expression of the EWS-FLI1 induced targets EZH2 and NR0B1 while inducing the expression of the repressed target PHLDA. Lower concentration (20 nM for 18 hours) of mithramycin had a minimal impact on expression of EWS-FLI1 induced (NR0B1, EZH2) or repressed targets (PHLDA1) unless combined with 2 μM PHA-767491. Data represents fold change (2ΔΔCT) in expression relative to GAPDH as measured by RT-qPCR in TC32 (n=6), TC252 (n=6), and TC71 (n=3) treated with either media (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C) for 18 hours. Each biological replicate had 3 or 4 technical replicates. * = P < 0.05, ** = P < 0.01, *** = P <0.001, **** = P < 0.0001, error bars show standard deviation. C, EWS-FLI1 downstream target proteins are suppressed with high dose mithramycin or the combination of mithramycin and PHA-767491. Immunoblot showing expression of the EWS-FLI1 downstream targets (NR0B1, EZH2) relative to loading control (GAPDH) following 18-hour exposure to medium (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C). Immunoblots shown are representative of three independent experiments per cell line. D and E, Reversal of the EWS-FLI1 gene signature requires either high dose mithramycin or combination treatment as demonstrated by RNA sequencing following treatment with medium (M), solvent (S), or 100 nM mithramycin (100 MMA), 20 nM mithramycin (20), 2 μM PHA-767491 (PHA), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (Combo) for 12 hours in TC32 (n=3) and TC252 (n=3) cell lines.

Article Snippet: The antibodies used in this study: EZH2 (D2C9) XP rabbit monoclonal antibody (1:1000 dilution; Cell Signaling), FLI1 (ab133485) rabbit monoclonal antibody (1:1000 dilution; Abcam), NR0B1/Dax1 (ab196649) rabbit monoclonal antibody (1:1000 dilution; Abcam), GAPDH (ab8245) mouse monoclonal antibody (1:500 dilution; Abcam), RNAPII CTD repeat YSPTPS (phospho S2) rabbit polyclonal antibody (1:1000 dilution, Abcam), RPB1 CTD (4H8) mouse monoclonal antibody (1:1000 dilution Cell Signaling).

Techniques: Activity Assay, Expressing, Concentration Assay, Quantitative RT-PCR, Standard Deviation, Western Blot, RNA Sequencing Assay

Journal: Molecular cancer therapeutics

Article Title: CDK9 Blockade Exploits Context-Dependent Transcriptional Changes to Improve Activity and Limit Toxicity of Mithramycin for Ewing Sarcoma

doi: 10.1158/1535-7163.MCT-19-0775

Figure Lengend Snippet: 2 μM PHA-767491 inhibits CDK9. A, 2 μM PHA-767491 blocks serine-2 phosphorylation independently or in combination with mithramycin. Immunoblot showing RNAPII and RNAPII CTD phosphoserine-2 relative to GAPDH loading control in TC32, TC252, and TC71 cell lines following exposure to medium (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C) for 18 hours. Data representative of three independent experiments. B, 2 μM PHA-767491 induces the expression of endogenous retroviral RNA (ERV). Data represents fold change in expression (2ΔΔCT) of ERV-F and ER9–1 relative to GAPDH in TC32 (n=3), TC252 (n=3), and TC71 (n=3) cells following exposure to medium (M), solvent (S), 100 nM mithramycin (100), 20 nM mithramycin (20), 2 μM PHA-767491 (P), or a combination of 20 nM mithramycin and 2 μM PHA-767491 (C) for 18 hours. C, Schematic of nuclear run on assay used to measure RNA processivity. Primer pairs to both a proximal and distal region on the EZH2 locus were used for RT-qPCR. D, Processivity of RNA as measured by qPCR enrichment of mRNA from the proximal (start) vs. distal (end) amplicon of EZH2 relative to solvent after a TC32 nuclear run-on assay. Nuclei were exposed to 2 μM PHA-767491 during the run-on reaction (PHA), 20 nM mithramycin during the run-on reaction (MMA Run on), or cells were pretreated with 20 nM mithramycin for 18 hours before the run-on reaction (MMA Pre). Each biological replicate in the figure had three technical replicates. * = P < 0.05, ** = P < 0.01, *** = P <0.001, **** = P < 0.0001, error bars show standard deviation.

Article Snippet: The antibodies used in this study: EZH2 (D2C9) XP rabbit monoclonal antibody (1:1000 dilution; Cell Signaling), FLI1 (ab133485) rabbit monoclonal antibody (1:1000 dilution; Abcam), NR0B1/Dax1 (ab196649) rabbit monoclonal antibody (1:1000 dilution; Abcam), GAPDH (ab8245) mouse monoclonal antibody (1:500 dilution; Abcam), RNAPII CTD repeat YSPTPS (phospho S2) rabbit polyclonal antibody (1:1000 dilution, Abcam), RPB1 CTD (4H8) mouse monoclonal antibody (1:1000 dilution Cell Signaling).

Techniques: Western Blot, Expressing, Nuclear Run-on Assay, Quantitative RT-PCR, Amplification, Standard Deviation