Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. wtEGFR and EGFRvIII bands are marked with * and **, respectively. ( b ) Densitometric quantification of galectin1 protein level normalized to tubulin in different BTSC lines is shown. ( c-d ) EGFR / EGFRvIII KD (si EGFR ) and control BTSCs (siCTL) were analyzed by immunoblotting as described in a. ( e-h ) BTSCs were treated with 1 or 5 µM lapatinib and galectin1 expression was assessed by immunoblotting (e-f) and immunostaining (g-h). Nuclei were stained with DAPI. Scale bar = 10 μm. ( i ) BTSCs were subjected to immunoblotting analysis using the antibodies indicated on the blots. ( j ) Pearson correlation analysis of pSTAT3-Y705 and galectin1 protein expression in different BTSCs is shown. ( k-l ) STAT3 KD (si STAT3 ) and siCTL BTSCs were analyzed by immunoblotting as described above. ( m-p ) BTSCs were subjected to immunoblotting or immunostaining following treatment with 25 or 50 µM of the STAT3 inhibitor, S3I-201. Scale bar = 10 μm. ( q-s ) EGFRvIII-expressing BTSCs were subjected to ChIP using an antibody to STAT3 or IgG control followed by qPCR using two different pairs of primers ( LGALS1 -a and LGALS1 -b). OSMR , and HPRT loci were used as positive and negative controls, respectively. ( t-u ) Luciferase reporter assay was performed in BTSC73 following KD of STAT3 using siRNA (t) or treatment with STAT3 inhibitors, 5 µM WP1066 or 50 μM S3I-201 (u). Data are presented as the mean□±□SEM, n ≥ 3. Unpaired two-tailed t -test (q, r and s); one-way ANOVA followed by Dunnett’s test (b) or Tukey’s test (t and u),*p < 0.05, **p < 0.01, ***p < 0.001. See also Figures S1 and S2.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Western Blot, Expressing, Immunostaining, Staining, Luciferase, Reporter Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) Cell viability was assessed by CellTiter-Glo assay in LGALS1 CRISPR and CTL BTSCs. ( c ) Population growth curves for LGALS1 CRISPR and CTL BTSC73 are shown. ( d-f ) Cell viability assay (d-e) and population growth curves (f) of BTSC73 treated with 1 or 10 µM OTX008 are shown. ( g ) Representative images of EdU staining in LGALS1 CRISPR and CTL BTSC73 are shown. ( h ) The number of EdU positive cells was quantified using Fiji software. ( i ) EdU incorporation was analyzed by flow cytometry in LGALS1 CRISPR and CTL BTSC73. Representative scatter plots of flow cytometry analyses are shown. Data are presented as the mean□±□SEM, n = 3. Unpaired two-tailed t -test (a, b, c and h); one-way ANOVA followed by Dunnett’s test (d, e and f), **p < 0.01, ***p < 0.001. See also Figures S3 and S4.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Glo Assay, CRISPR, Viability Assay, Staining, Software, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-b ) LGALS1 CRISPR or CTL BTSC73 were subcutaneously injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (a). Graph represents tumour mass (b). ( c-f ) BTSC73 or BTSC147 were injected subcutaneously into SCID mice and treated with 10 mg/kg OTX008. Representative bioluminescence real-time images tracing tumour growth are shown (c, e). Graphs represent tumour mass (d, f). ( g-j ) LGALS1 CRISPR or CTL BTSC73 were intracranially injected into SCID mice. Representative bioluminescence real-time images tracing tumour growth are shown (g). Intensities of luciferase signal were quantified at different time points using Xenogen IVIS software (h). Graph represents quantification of animal weight (i). KM survival plot was graphed to evaluate mice lifespan in each group (j). Data are presented as the mean□±μSEM, n ≥ 4 mice. Unpaired two-tailed t -test (b, d, f, h and i); log-rank test (j), **p < 0.01, ***p < 0.001.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Injection, Luciferase, Software, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) Volcano plot representing LGALS1 differentially regulated genes is shown. ( b-c ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal (b) and proneural (c) subtypes of glioblastoma. ( d ) GSEA analysis demonstrates enrichment for gene sets corresponding to mesenchymal-like meta-module (MES1-like) signature. ( e-f ) GSEA analysis demonstrates enrichment for gene sets corresponding to recruitment of NuMA to mitotic centrosomes (e) and mitotic G2−G2/M phases (f). ( g-h ) RNA-seq data was validated by RT-qPCR in BTSC73 and BTSC147. ( i-j ) Cell cycle distribution was assessed by flow cytometry after PI staining in LGALS1 CRISPR BTSCs. Data are presented as the mean□±□SEM, n = 3. One-way ANOVA followed by Dunnett’s test (g and h); unpaired two- tailed t -test (i and j), *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S5.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: RNA Sequencing Assay, Quantitative RT-PCR, Flow Cytometry, Staining, CRISPR, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a-d ) LGALS1 CRISPR and CTL EGFRvIII-expressing BTSCs were subjected to LDA (a-b) or ELDA (c-d). ( e-f ) EGFRvIII-expressing LGALS1 CRISPR and CTL BTSCs were subjected to clonogenicity assay performed by culturing one single cell per well. ( g-h ) BTSCs that don’t harbour the EGFRvIII mutation were electroporated with siCTL or si LGALS1 and subjected for ELDA analysis. ( i-p ) EGFRvIII-expressing BTSCs were subjected to LDA (i, j, m and n) or ELDA (k, l, o and p) following the treatment with 1 or 10 µM OTX008. ( q-t ) BTSCs that don’t harbour the EGFRvIII mutation were subjected to LDA (q-r) or ELDA (s-t) following the treatment with 1 or 10 µM OTX008. *p < 0.05, **p < 0.01, ***p < 0.001; unpaired two-tailed t -test (a, b, e and f); one-way ANOVA followed by Dunnett’s test (i, j, m and n), n = 3. Data are presented as the mean□±□SEM. See also Figure S6.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Expressing, Mutagenesis, Two Tailed Test

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) ELDA was performed following 4 Gy of IR in LGALS1 CRISPR or CTL BTSCs. ( b-c ) LGALS1 CRISPR and CTL BTSC73 were subjected to IR (8□Gy). Apoptosis analysis was performed by flow cytometry 48□h following IR using annexin V and PI double staining. Representative scatter plots of flow cytometry analyses are shown (b). The percentage of cell death (annexin V positive cells) is presented in the histogram (c), n□=□3. ( d ) Schematic diagram of the experimental procedure is shown. BTSC73 were intracranially injected into SCID mice and then treated with OTX008, 4□Gy of IR or a combination of OTX008 and IR. ( e ) Representative bioluminescence real-time images tracing tumour growth are shown, n□=□6 mice. ( f ) Coronal sections of mouse brains were stained with hematoxylin and eosin on day 22 after injection. Representative images of 3 different tumour sections are shown. Scale bar = 1□mm, scale bar (inset) = 0.2 mm. ( g ) Intensities of luciferase signal were quantified at different time points, n = 6 mice. ( h ) KM survival plot was graphed to assess animal lifespan, n□=□6 mice. ( i ) Survival extension of mice bearing BTSC-derived tumours treated with OTX008, IR, or OTX008 + IR relative to those treated with the vehicle control. Data are presented as the mean□±□SEM. One-way ANOVA followed by Tukey’s test (c and i); log-rank test (h), *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: CRISPR, Flow Cytometry, Double Staining, Injection, Staining, Luciferase, Derivative Assay

Journal: bioRxiv

Article Title: Transcriptional Control of Brain Tumour Stem Cells by a Carbohydrate Binding Protein

doi: 10.1101/2021.04.14.439704

Figure Lengend Snippet: ( a ) LGALS1 -differentially regulated genes were subjected to enrichment analysis of TF binding motifs using oPOSSUM-3 software. ( b ) Volcano plot representing the HOXA5 target genes among the LGALS1 -differentially-regulated genes is shown. ( c ) BTSCs were analyzed by immunoblotting using the antibodies indicated on the blots. ( d ) Pearson correlation analysis of HOXA5 and galectin1 protein expression is shown. ( e ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients is shown. ( f ) Relative positions of HOXA5 ChIP-seq peaks to the adjacent TSS of LGALS1 -differentially regulated genes are shown. The x-axis indicates the distance between peak centers and the TSS of adjacent LGALS1 -differentially regulated genes. The y-axis denotes the expression ratios (log2) of the LGALS1 -differentially regulated gene. Circle size indicates HOXA5 peak height, and color denotes the conservation score of HOXA5 peaks. ( g-h ) HOXA5 KD (si HOXA5 ) and siCTL BTSCs were subjected to RT-qPCR analysis. ( i ) ELDA was performed following 4LGy of IR in si HOXA5 vs. siCTL. ( j - m ) Endogenous Co-IP experiments were performed in different BTSC lines using an anti-HOXA5 antibody, followed by immunoblotting with galectin1 and HOXA5 antibodies. ( n ) Co-IP experiment was performed using anti-FLAG antibody, followed by immunoblotting with anti-FLAG and anti-HOXA5 antibodies. ( o - r ) PLA of galectin1 and HOXA5 were performed in different BTSC lines. Primary antibodies were omitted for the controls. Nuclei were stained with DAPI. Scale bar = 10 μm. ( s ) LGALS1 CRISPR and CTL BTSC73 were subjected to ChIP using an antibody to HOXA5 followed by qPCR for HOXA5 candidate target genes. HBB locus was used as a negative control. ( t-u ) KM survival plot describing the association between LGALS1 and HOXA5 expression and the survival of glioblastoma patients treated with radiotherapy (microarray G4502A Agilent, level 3, n = 489). Data are presented as the meanL±LSEM, n = 3. Log-rank test (e, t and u); one-way ANOVA followed by Dunnett’s test (g and h); unpaired two-tailed t -test (s). *p < 0.05, **p < 0.01, ***p < 0.001. See also Figure S7.

Article Snippet: The upstream 376 bp region of the human LGALS1 transcriptional start site was cloned into the pGL4.23 (Promega) vector to generate the LGALS1 luciferase reporter gene ( LGALS1 pGL4.23) by digesting the plasmid and the annealed primer pair using EcoRV (NEB, #R0195L) and HindIII (NEB, #R0104L) and ligating them with T4 DNA ligase (NEB, #M0202L).

Techniques: Binding Assay, Software, Western Blot, Expressing, ChIP-sequencing, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Staining, CRISPR, Negative Control, Microarray, Two Tailed Test

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: RNA Immunoprecipitation, Purification, Negative Control, Sequencing, Hybridization, 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, Expressing, Microarray, ChIP-sequencing, 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) 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, Control, 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: 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, ChIP-sequencing, Gene Expression, Binding Assay, Incubation, Control, ChIP-qPCR

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: Control, 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, Control, Migration, Injection, Staining, Activity Assay, Blocking Assay, Expressing, Inhibition

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Global analyses of IKZF1 and c‐FOS binding to the myeloma genome. (A) Upper panel: Average plot (middle) and heatmap (left) of IKZF1 chromatin immunoprecipitation (ChIP)‐seq reads over all transcription start sites (TSSs) ± 5000 bp. The pie chart shows the gene section breakdown (right). Lower panel: Average plot (middle) and heatmap (left) of c‐FOS ChIP‐seq reads over all TSSs ± 5000 bp. The pie chart shows the gene section breakdown (right). Genes (rows) were ordered in the same way in heatmaps. (B) Left panel: Overlap of IKZF1‐ ( n = 13 932) and c‐FOS‐binding ( n = 10 173) sites in MM.1S cells. Right panel: Status of IKZF1 and c‐FOS binding at promoter/enhancer regions of representative genes based on ChIP‐seq data. (C) Nucleotide sequences of IKZF1‐ and c‐FOS‐binding sites deduced from ChIP‐seq analyses of MM.1S cells. The ChIP‐seq data were analyzed using a Partek Flow genomic analysis software v10.0 (Partek Inc.).

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Binding Assay, Chromatin Immunoprecipitation, ChIP-sequencing, Software

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: c‐FOS‐binding sites are present in nearly one‐half of IKZF1‐target genes in multiple myeloma (MM). (A) Using chromatin immunoprecipitation (ChIP)‐seq data of MM.1S cells, we visualized IKZF1 binding near transcription start sites (TSSs) (red triangles) of the indicated genes in the UCSC genome browser. The genes possessing the activator protein‐1 (AP‐1)‐binding motif based on a TRANSFAC search ( https://genexplain.com/transfac/ ) are marked in red, and those without the conventional AP‐1‐binding motif are marked in blue. (B) The peak values of IKZF1 binding and the presence of AP‐1‐binding sites in selected genes. (C) An example of the co‐occupancy of IKZF1 and c‐FOS in MM.1S cells.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Binding Assay, Chromatin Immunoprecipitation, ChIP-sequencing

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Co‐occupancy of IKZF1 and c‐FOS at promoter/enhancer regions of actively transcribed genes in multiple myeloma (MM). Upper panel: chromatin immunoprecipitation (ChIP)‐seq data of IKZF1 and c‐FOS binding in MM.1S cells were aligned with acetylated histone H3K27 marks in the UCSC genome browser. The transcription start site (TSS) of each gene is shown with red triangles. Lower panel: Gene expression was assayed using Affymetrix U133 plus 2.0 microarrays. The data are unpaired GCRMA‐normalized expression signals for each gene in CD138‐positive cells from patients with (1) monoclonal gammopathy of undetermined significance, (2) newly‐diagnosed MM and (3) plasma cell leukaemia ( n = 8 each). ⁵³ (A) The results of three representative genes that are highly expressed in plasma cell disorders. (B) The results of three representative genes not expressed in plasma cell disorders.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Chromatin Immunoprecipitation, ChIP-sequencing, Binding Assay, Gene Expression, Expressing, Clinical Proteomics

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Biological functions of activator protein‐1 (AP‐1) family proteins in multiple myeloma (MM). (A) We determined the correlation of expression levels between IKZF1‐target genes (y‐axis) and the indicated genes (x‐axis) using the GenomicScape tool ( http://www.genomicscape.com ). ³² The data were extracted from DNA microarray analyses of gene expression in newly‐diagnosed MM patients. ⁵³ (B) Cell lysates were prepared from the indicated MM cell lines and analyzed by immunoblotting using antibodies against c‐FOS, FOSB, c‐JUN and GAPDH (loading control). (C) We examined the expression of FOS , FOSB and JUN transcripts in normal plasma cells ( n = 22), CD138‐positive cells derived from patients with monoclonal gammopathy of undetermined significance (MGUS; n = 44) and MM cells from newly‐diagnosed patients ( n = 12). The data were extracted from DNA microarray analyses deposited in the GenomicScape database by the Arkansas group. ⁵³ (D) Left panel: We transduced the indicated MM cell lines with shRNA against FOS or scrambled sequences (sh‐control) and evaluated the expression of candidate IKZF1‐target genes ( IRF4 , SLAMF7 , BSG and CD48 ) along with GAPDH (Control) and FOS (for the evaluation of knockdown efficiency) using quantitative real‐time reverse transcription‐PCR after 24 h. The data were quantified by the 2 –∆∆Ct method using GAPDH as a reference and are shown as the fold changes against the values of sh‐control‐transfected cells. * p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. Right panel: Correlation between the expression levels of FOS mRNA and those of IRF4 or CD48 mRNA in MM cell lines.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Expressing, Microarray, Gene Expression, Western Blot, Control, Clinical Proteomics, Derivative Assay, shRNA, Knockdown, Reverse Transcription, Transfection, Comparison

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Direct interaction of IKZF1 and c‐FOS in multiple myeloma (MM) cells. (A) Left panel: Nuclear extracts from MM.1S and KMS12‐BM cells were immunoprecipitated with rabbit anti‐IKZF1 antibody or isotype‐matched immunoglobulin (IgG). The immunoprecipitates were analyzed by immunoblotting with specific antibodies against IKZF1, IKZF3, c‐FOS, c‐JUN, IRF4 and rabbit IgG. Input: Immunoblotting of nuclear extracts fractioned before immunoprecipitation. Right panel: The same experiments were carried out with c‐FOS immunoprecipitates. (B) The binding of the IKZF1 complex to oligonucleotides containing an IKZF consensus motif was measured by sandwich immunoassay and is shown as the relative activity against the data obtained in the absence of blocking antibodies. Antibody perturbation was carried out with isotype‐matched immunoglobulin (Control), an anti‐IKZF1 antibody, an anti‐c‐FOS antibody and a combination of the two antibodies. p < .05 by Student's t ‐test against Control ( n = 5). (C) HEK293T cells were transfected with an empty vector (Mock) or expression vectors carrying HA‐tagged full‐length IKZF1 protein, the exon 1‐exon 4 fragment, the exon 5‐exon 6 fragment, or the exon 7 fragment of IKZF1 together with a FLAG‐tagged c‐FOS expression vector. Nuclear extracts were isolated 24 h after transfection and immunoprecipitated with an anti‐HA antibody, followed by immunoblotting with antibodies against HA tag (IKZF1), FLAG tag (c‐FOS) or rabbit immunoglobulin (IgG). Red arrows denote the positions of the transfected IKZF1 fragments. (D) Structure‐based prediction of IKZF1‐c‐FOS interactions using the AlphaFold2 program.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Activity Assay, Blocking Assay, Control, Transfection, Plasmid Preparation, Expressing, Isolation, FLAG-tag

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: c‐FOS mediates lenalidomide resistance in multiple myeloma (MM) cells. (A) Left panel: MM.1S and KMS12‐BM cells were transfected with c‐FOS expression vector or empty vector (Mock) and treated with the vehicle alone (None) or 2.5 μM lenalidomide for 24 h, followed by immunoblot analysis for the expression of the indicated molecules. Right panel: MM.1S and KMS12‐BM cells were transfected with a c‐FOS expression vector or an empty vector (Mock) and treated with various concentrations of lenalidomide for 72 hours. Cell viability was determined by MTT reduction assay with a Cell Counting Kit (Fujifilm Wako Biochemicals). The graphs show the means of triplicate samples; the S.D. was less than 10% and thus omitted. * p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. (B) Upper panel: Schematic representation of the IRF4 promoter region from the chromatin immunoprecipitation (ChIP)‐Atlas data. The relative positions of the putative binding sites of transcription factors are approximated by the symbols shown in the box. TSS: transcription start site. Bidirectional red arrows indicate regions that were PCR amplified in ChIP assays. Lower panel: Chromatin suspensions were prepared from KMS12‐BM cells cultured with vehicle alone (DMSO) or 2.5 μM lenalidomide for 24 h and immunoprecipitated with anti‐IKZF1 (grey bars) and c‐FOS (pink bars) antibodies or IgG (back bars). The resulting precipitates were subjected to PCR to amplify the regions shown in the upper panel. * p < .05 against IgG by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests. (C) The expression of IRF4 protein and mRNA in DMSO‐ or lenalidomide‐treated KMS12‐BM cells. (D) MM.1S and KMS12‐BM cells were transfected with sh‐FOS expression vector or empty vector (Control) and treated with vehicle alone (DMSO) or 10 μM lenalidomide. (E) MM.1S and KMS12‐BM cells were treated with vehicle alone (DMSO), 10 μM lenalidomide, 20 μM T‐5224, or the combination of lenalidomide and T‐5224. Upper panels: The expressions of IRF4 and GAPDH transcripts were examined by quantitative real‐time reverse transcription‐PCR after 24 h. The results were normalized to the values of DMSO‐treated cells. Lower panels: Cell viability was determined by MTT reduction assay after 72 h and is shown as the percentage of untreated cells (%Control). The data are presented as the means of three biological replicates with S.D. (bars). * p < .05 by one‐way ANOVA with Student–Newman–Keuls multiple comparison tests.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, MTT Reduction Assay, Cell Counting, Comparison, Chromatin Immunoprecipitation, Binding Assay, Amplification, Cell Culture, Immunoprecipitation, Control, Reverse Transcription

Journal: Clinical and Translational Medicine

Article Title: c‐FOS is an integral component of the IKZF1 transactivator complex and mediates lenalidomide resistance in multiple myeloma

doi: 10.1002/ctm2.1364

Figure Lengend Snippet: Graphical abstract. c‐FOS, a subunit of the activator protein‐1 (AP‐1) transactivator, is an integral component of the IKZF1 complex and is primarily responsible for the activator function of the complex in multiple myeloma (MM) cells. Left panel: The IKZF complex binds to the enhancer/promoter regions of the genes involved in the growth and survival of MM cells such as IRF4 and SLAMF7 through the canonical IKZF‐binding motif CTTCC with c‐FOS/c‐JUN. Middle panel: Lenalidomide induces ubiquitin‐dependent degradation of IKZF1/IKZF3; however, residual c‐FOS, the level of which is often increased by lenalidomide treatment, binds to the AP‐1 consensus sequences, which present in the vicinity of IKZF‐binding sites of certain genes including IRF4 and SLAMF7 , leading to sustained expression of these genes and lenalidomide resistance. Right panel: A selective AP‐1 inhibitor, T‐5224, binds to the DNA‐binding domain of c‐FOS and mitigates the residual activity of the MM‐specific activator complex, resulting in complete IRF4 down‐regulation and augmentation of the anti‐MM effects of lenalidomide.

Article Snippet: For immunoprecipitation, we incubated the primary antibody with protein A magnetic beads (Thermo Fisher Scientific) at 4°C for 24 h, added cell lysates to the antibody‐bound beads in solution after discarding the supernatants, and rotated the samples for 30 min. After washing, immunoprecipitates were eluted and subjected to SDS‐PAGE, followed by immunoblotting using antibodies against IKZF1 (#14859), IKZF3 (#15103), c‐FOS (#2250), FOSB (#2251), c‐JUN (#9165) (Cell Signaling Technology, Beverly, MA), IRF4 (sc‐48338), GAPDH (sc‐47724) (Santa Cruz Biotechnology), normal rabbit IgG (PM035) and FLAG tag (PM020) (MBL).

Techniques: Binding Assay, Ubiquitin Proteomics, Expressing, Activity Assay

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 3. HDAC3 is required for extracellular matrix homeostasis and remodeling of semilunar valves. A, Movat’s pentachrome staining of remodeling aortic valve shows an increase in proteoglycans (arrows, blue) from E13.5 to E18.5 in Hdac3Isl1KO aortic valves with a reduction and remodeling of proteoglycans in control valves. B, Movat’s pentachrome staining of remodeling pulmonic valve shows an increase in proteoglycans (arrows, blue) from E13.5 to E18.5 in Hdac3Isl1KO pulmonic valves with a reduction and remodeling of proteoglycans in control valves. C, Masson’s trichrome staining demonstrates disorganized collagen expression (arrow, blue) in Hdac3Isl1KO E18.5 aortic valve. D, Masson’s trichrome staining demonstrates disorganized collagen expression (arrow, blue) in Hdac3Isl1KO E18.5 pulmonic valve. E, Verhoeff-Van Gieson (VVG) staining shows disorganized collagen expression (arrow, red) in Hdac3Isl1KO E18.5 aortic valve. F, Verhoeff-Van Gieson staining shows disorganized collagen expression (arrow, red) in Hdac3Isl1KO E18.5 aortic valve. G, Movat’s pentachrome-stained sections showincreasedproteoglycans(blue)inaorticvalvecuspsofE18.5Hdac3Mef2CKOhearts.H,Movat’spentachromestainingrevealsincreasedproteoglycans(blue) in E18.5 Hdac3Mef2CKO pulmonic valve. I, representative images of cleaved caspase-3 immunostaining in E13.5 semilunar valves. Arrows, positive staining. J, quantification of cleaved caspase-3-positive cells in E13.5 control and Hdac3Isl1KO aortic valves. K, quantification of cleaved caspase-3-positive cells in E13.5 control and Hdac3Isl1KO pulmonic valves. L, schematic model depicting disorganized extracellular matrix and reduced apoptosis in Hdac3Isl1KO E13.5 semilunar valves. M, schematic model showing hyperplastic, enlarged, and disorganized Hdac3Isl1KO E18.5 semilunar valves.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Staining, Control, Expressing, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 4. HDAC3 is a critical regulator of TGF- signaling pathway. A–E, IPA of microarray data from E9.5 Hdac3Isl1KO hearts. A, cardiovascular development and function subcategories significantly dysregulated in E9.5 Hdac3Isl1KO hearts, sorted by significance, from IPA diseases and function analysis. B, classes of congenital heart anomalies affected in E9.5 Hdac3Isl1KO hearts, sorted by significance, from IPA diseases and function analysis. C, 30 most significant upstream regulatorsfromIPAupstreamanalysisofE9.5Hdac3Isl1KOmicroarraydata.D,clusteredheatmapofdifferentiallyexpressedgenesrelatedtoupstreamregulator TGF-1 from IPA upstream analysis of Hdac3Isl1KO microarray data. E, top 30 upstream regulators, based on number of associated genes differentially expressed in E9.5 Hdac3Isl1KO hearts from IPA upstream analysis. F, transcripts for Sumo1, Nrp2, Smad4, Snai1, Tgf-1, and Kpnb1 were detected by real-time qPCR in Hdac3F/F and Hdac3Isl1KO outflow tract with right ventricle derived from E9.5 embryos (mean S.E. (error bars), n 3). G–J, ChIP-qPCR analysis of HDAC3 recruitment to promoter-proximal regions of TGF- pathway genes performed in wild-type E9.5 outflow tract (mean S.E., n 3). K and L, ELISA for TGF-1 (K) and phospho-SMAD2/3 (L) was performed in Hdac3F/F and Hdac3Isl1KO outflow tracts (mean S.E., n 3 (K) and n 4 (L)). M, phospho-SMAD2/3 immunostaining (arrows) in E9.5 Hdac3Isl1KO; R26R-LacZ/ hearts.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Microarray, Derivative Assay, ChIP-qPCR, Enzyme-linked Immunosorbent Assay, Immunostaining

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 8. HDAC3 epigenetically silences TGF-1 within valvular mesenchymal cells by recruiting PRC2 complex to the NCOR complex. A, relative mRNA levels of TGF-1 in isolated cardiac endothelial cells (EC) or cardiac mesenchymal cells (MC) from E10.5 Hdac3F/F outflow tract cushion explants, infected either with CRE or GFP control lentivirus. B, relative mRNA levels of TGF-1 in isolated cardiac endothelial cells or dissected semilunar valves (SL) derived from Hdac3Isl1KO and Hdac3F/F E14.5 hearts. C, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in isolated cardiac endothelial cells or cardiac mesen- chymal cells from E10.5 outflow tract cushion explants. D, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in isolated cardiac endothelial cells or dissected semilunar valves from E14.5 hearts. E–K, ChIP-qPCR analysis of H3K27 trimethylation (E), H3K27 acetylation (F), RNA polymerase II (G), CREBBP (H), EZH2 (I), EED (J), and SUZ12 (K) upstream of TGF-1 in Hdac3Isl1KO and control E14.5 semilunar valves. L, ChIP-qPCR analysis of HDAC3 occupancy upstream of TGF-1 in E14.5 valvular mesenchymal cells infected with either control shRNA (sc-shRNA), EZH2 shRNA, or NCOR1 shRNA. M, ChIP-qPCR analysis of NCOR1 upstream of TGF-1 in Hdac3Isl1KO and control E14.5 semilunar valves. N, Co-ChIP for HDAC3 and either EZH2, NCOR1, H3K27me3, H3K27ac, or polymerase II upstream of TGF-1 in E14.5 wild-type dissected semilunar valves. O, total lysates from E14.5 wild-type pooled semilunar valves were immunoprecipitated (IP) by EZH2 antibody, and Western blot was performed using HDAC3 antibody. -Tubulin is shown as an input control. HDAC3 was quantified and normalized to total input -tubulin using ImageJ software (mean S.E. (error bars), n 3). P, ChIP-qPCR analysis of H3K27me3 upstream of TGF-1 in E14.5 valvular mesenchymal cells infected with either control shRNA, EZH2 shRNA, or NCOR1 shRNA.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Isolation, Infection, Control, Derivative Assay, ChIP-qPCR, shRNA, Immunoprecipitation, Western Blot, Software

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 9. HDAC3 functions in a deacetylase-independent manner to regulate EndMT and epigenetic silencing of TGF-1. A, HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression constructs were transfected in HEK-293T cells. Expression was detected by Western blot from whole cell lysates using FLAG antibody. GAPDH is shown as a loading control. B, HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression was quantified and normalized to total input GAPDH using ImageJ software (mean S.D. (error bars), n 3). C, HDAC activity of HDAC3-FLAG and HDAC3H134A,H135A-FLAG expression was quantified against a pseudosubstrate. D, EndMT assay of control- or Cre-infected E10.5 Hdac3F/F outflow tract cushion explants co-infected with GFP, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses, imaged 24 h after isolation. E, quantification of average radial migration, measured in eight directions, of control- or Cre-infected E10.5 Hdac3F/F outflow tract cushion explants co-infected with GFP, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses, measured 24 h after isolation(mean S.E.(errorbars),n3).F,relativemRNAlevelsofTgf-1incontrol-orCre-infectedE14.5Hdac3F/Fvalvularmesenchymalcellsco-infectedwith control, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses (mean S.E., n 3). G, a 1309-bp TGF-1 promoter luciferase reporter (WT) or a truncated, 1267-bp TGF-1 promoter luciferase reporter, lacking an HDAC3-enriched region (mutant) were transfected in murine endothelial cells with and without an HDAC3-FLAG or HDAC3H134A,H135A-FLAG expression plasmid. Induction is represented as a ratio of firefly and Renilla luciferase activity. H–L, ChIP-qPCR analysis of H3K27 acetylation (H), H3K27 trimethylation (I), EZH2 (J), EED (K), and SUZ12 (L) upstream of TGF-1 in control- or Cre-infected E14.5 Hdac3F/F valvular mesenchymal cells co-infected with control, HDAC3-FLAG, or HDAC3H134A,H135A-FLAG lentiviruses (mean S.E., n 3).

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Histone Deacetylase Assay, Expressing, Construct, Transfection, Western Blot, Control, Software, Activity Assay, Infection, Isolation, Migration, Luciferase, Mutagenesis, Plasmid Preparation, ChIP-qPCR

Journal: Journal of Biological Chemistry

Article Title: Histone Deacetylase 3 Coordinates Deacetylase-independent Epigenetic Silencing of Transforming Growth Factor-β1 (TGF-β1) to Orchestrate Second Heart Field Development

doi: 10.1074/jbc.m115.684753

Figure Lengend Snippet: FIGURE 10. Summary of phenotypes and proposed model of HDAC3 function within second heart field progenitor cells and second heart field-derived mesenchymal cells. A, loss of HDAC3 in second heart field progenitor cells leads to outflow tract and semilunar valve pathologies. Strikingly, genetic deletion of HDAC3 in differentiated mesenchymal and smooth muscle cells (Hdac3TaglnKO) recapitulates the majority of these phenotypes. However, deletion of HDAC3 in differentiated cardiomyocytes (Hdac3Myh6KO) or endothelial cells (Hdac3Cdh5KO) did not recapitulate the cardiovascular defects observed in Hdac3Isl1KO embryos. B, Hdac3Isl1KO hearts exhibit disorganized collagen and elastin within dilated aortic walls and hyperplastic semilunar valves containing activated myofibroblasts and disorganized extracellular matrix. In both control and Hdac3Isl1KO cardiac endothelial cells, the upstream regulatory region of TGF-1 is occupied by RNA polymerase II and CREBBP and exhibits H3K27 acetylation concomitant with TGF-1 expression. In control semilunar valves, endothelial cells undergo EndMT to become mesen- chymalcells.Inthesemesenchymalcells,NCOR1,HDAC3,andPRC2complex(EZH2,EED,andSUZ12)arerecruitedtotheupstreamregulatoryregionofTGF-1,which becomes trimethylated on histone H3 Lys-27, and TGF-1 expression is epigenetically silenced. In Hdac3Isl1KO hearts, EZH2, EED, and SUZ12 are not recruited to the TGF-1 regulatory region, RNA polymerase II and CREBBP are present, and histone H3 Lys-27 remains acetylated, favoring aberrant expression of TGF-1 in mesen- chymal cells. TGF-1 activates mesenchymal cells to become myofibroblasts, which perpetuate EndMT and activation of mesenchymal cells through continued induction of TGF-1 and aberrant expression of extracellular matrix, including proteoglycans and collagen.

Article Snippet: Antibodies and Reagents—The following antibodies were used in this study: HDAC3 (Abcam and Santa Cruz Biotechnology), phospho-HDAC3 (Ser-424) (Cell Signaling), TGF- panspecific polyclonal antibody (R&D Systems), SMAD2/3 (Santa Cruz Biotechnology), phospho-SMAD2/3 (Ser-423/425) (Santa Cruz Biotechnology), vimentin (Santa Cruz Biotechnology), PECAM1 (BD Pharmingen), troponin T (Developmental Studies Hybridoma Bank, Iowa City, IA), MF-20 (Developmental Studies Hybridoma Bank, Iowa City, IA), cleaved caspase-3 (Cell Signaling), RNA polymerase II (Abcam), EZH2 (Abcam), NCOR1 (Abcam), H3K27ac (Abcam), H3K27me3 (Abcam), EED (Abcam), SUZ12 (Abcam), CREBBP (Abcam), IgG (R&D Systems), GAPDH (R&D Systems), FLAG (Sigma), -tubulin (Sigma), IRDye-conjugated secondary antibodies (LI-COR), Alexa Fluor 546-conjugated secondary antibody (Life Technologies), and biotinylated universal pan-specific antibody 27068 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 290 • NUMBER 45 • NOVEMBER 6, 2015 at U N IV O F N E B R A SK A - L incoln on M ay 31, 2016 http://w w w .jbc.org/ D ow nloaded from (horse anti-mouse/rabbit/goat IgG) (Vector Laboratories).

Techniques: Derivative Assay, Control, Expressing, Activation Assay

Journal: Cell Reports Medicine

Article Title: LTA4H improves the tumor microenvironment and prevents HCC progression via targeting the HNRNPA1/LTBP1/TGF-β axis

doi: 10.1016/j.xcrm.2025.102000

Figure Lengend Snippet:

Article Snippet: Anti-Histone H3 , Bioss , BSM-33042M.

Techniques: Microarray, Recombinant, Lysis, Protease Inhibitor, SYBR Green Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Reverse Transcription, Activity Assay, Immunoprecipitation, Extraction, Purification, In Vitro, Transfection, Sequencing, Mass Cytometry, ChIP-sequencing, Transgenic Assay, Software