Journal: Epigenetics & chromatin

Article Title: Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity.

doi: 10.1186/s13072-022-00461-8

Figure Lengend Snippet: Fig. 1 Selection of MEN1 missense mutation-derived forms of menin. A Nine disease-relevant MEN1 missense mutations were selected based on their predicted stability in silico and ability to bind to MLL1 and/or JunD. The predicted effects of the mutations are listed. B The nine MEN1 mutations are projected on the menin structure

Article Snippet: To localize MLL1 or RNA polymerase II standard CUT&RUN [32], was employed using MLL1/KMT2A antibodies (Epicypher #SKU 13–2004) or 8WG16 antibodies directed against the CTD of RPB1 at 1 μg/ml.

Techniques: Selection, Mutagenesis, Derivative Assay, In Silico

Journal: Epigenetics & chromatin

Article Title: Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity.

doi: 10.1186/s13072-022-00461-8

Figure Lengend Snippet: Fig. 6 Genome-wide binding events of the menin mutants. A Correlation of the coverage between WT menin and menin mutants. Values represent Pearson correlations of normalized coverage calculated for 10-kb bins. B Coverage tracks of the regions bound by menin WT and menin mutants. The left box highlights a genomic region occupied by both menin WT and mutant proteins, while the right box highlights a genomic region where binding of the menin mutants R52G, E255K and E359K was lost. C Percentages of peaks retained at menin WT bound sites by greenCUT&RUN mapping using the indicated menin mutant. In blue, retained binding at menin–JunD sites without MLL1/H3K4me3 is shown. In red, retained binding of mutant forms of menin at WT menin–MLL1/H3K4me3 sites without JunD is shown. D Heatmaps showing coverage on the retained (upper panel) and lost (lower panel) peaks with menin mutants relative to WT menin. The number of the peaks are given on the left side. The coverage is shown on the top of each heatmaps. Please note that the E408Q mutant essentially behaves like WT menin

Article Snippet: To localize MLL1 or RNA polymerase II standard CUT&RUN [32], was employed using MLL1/KMT2A antibodies (Epicypher #SKU 13–2004) or 8WG16 antibodies directed against the CTD of RPB1 at 1 μg/ml.

Techniques: Genome Wide, Binding Assay, Mutagenesis

Journal: Epigenetics & chromatin

Article Title: Multi-omics analyses of MEN1 missense mutations identify disruption of menin-MLL and menin-JunD interactions as critical requirements for molecular pathogenicity.

doi: 10.1186/s13072-022-00461-8

Figure Lengend Snippet: Fig. 7 Projection of the selected MEN1 gene mutations menin R52G, E255K, E359K and E408Q (in green) on the tertiary structure of WT menin. Overlapping positions of the MLL1 peptide (in brown) and of the JunD peptide (in blue) are indicated

Article Snippet: To localize MLL1 or RNA polymerase II standard CUT&RUN [32], was employed using MLL1/KMT2A antibodies (Epicypher #SKU 13–2004) or 8WG16 antibodies directed against the CTD of RPB1 at 1 μg/ml.

Techniques:

Journal: Pathology, research and practice

Article Title: WDR5 drives the development of cervical squamous cell carcinoma by inducing epithelial-mesenchymal transition and cancer-associated fibroblasts formation.

doi: 10.1016/j.prp.2022.154076

Figure Lengend Snippet: Fig. 5. CXCL8 is regulated by the interaction between WDR5 and MLL1. (A and C) Expression of CXCL8 in CAFs was down-regulated after WDR5 knock-down, while the knock-down of WDR5 had no impact on CXCL8 expression when MM102 was used, and MM102 markedly inhibited CXCL8 expression in CAFs. (B and D) Expression of CXCL8 in hCECs was up-regulated after WDR5 overexpression, while the overexpression of WDR5 had no impact on CXCL8 expression when MM102 was used. (E and F) ChIP analysis revealed that the distributions of WDR5, MLL1, and H3K4me3 in CXCL8 promoter were overlapping, and the recruitments of WDR5 and MLL1 in CXCL8 promoter were significantly higher in CAFs than that in hCECs, as well as the H3K4me3 level in this region. Results were obtained from at least three independent experiments. Data were shown as mean ± SEM and were analyzed by student’s t-test (two groups) or one-way ANOVA (multiple groups). * * P < 0.01, * ** P < 0.001.

Article Snippet: Chromatin was immunoprecipitated using antibodies against WDR5 (13105, Cell Signaling Technology, USA), H3K4me3 (ab8580, Abcam, USA), and MLL1 (34907, Cell Signaling Technology, USA).

Techniques: Expressing, Knockdown, Over Expression

Journal: Pathology, research and practice

Article Title: WDR5 drives the development of cervical squamous cell carcinoma by inducing epithelial-mesenchymal transition and cancer-associated fibroblasts formation.

doi: 10.1016/j.prp.2022.154076

Figure Lengend Snippet: Fig. 6. Blocking the interaction between WDR5 and MLL1 suppresses CC development. (A) Using MM102 significantly suppressed tumor growth. (B and C) Using MM102 (10 ug/g) markedly down-regulated the expressions of CXCL8 and FAP in tumor lesions. (D) Using MM102 (10 ug/g) markedly changed the expression levels of EMT markers. (E) Using MM102 (10 ug/g) significantly down-regulated cytokines secretions in tumor lesions. Results were obtained from at least three inde pendent experiments. Ten mice in each group. Data were shown as mean ± SEM and were analyzed by student’s t-test (two groups) or one-way ANOVA (multiple groups). * * P < 0.01, * ** P < 0.001.

Article Snippet: Chromatin was immunoprecipitated using antibodies against WDR5 (13105, Cell Signaling Technology, USA), H3K4me3 (ab8580, Abcam, USA), and MLL1 (34907, Cell Signaling Technology, USA).

Techniques: Blocking Assay, Expressing

Journal: Pathology, research and practice

Article Title: WDR5 drives the development of cervical squamous cell carcinoma by inducing epithelial-mesenchymal transition and cancer-associated fibroblasts formation.

doi: 10.1016/j.prp.2022.154076

Figure Lengend Snippet: Fig. 7. Overview of the mechanism of WDR5 promoting CSCC development. WDR5 promotes the EMT process and enhances the transformation of CAFs by interacting with MLL1 and inducing CXCL8 expression. CAFs secret cytokines such as GM-CSF, IL-6, CCL2, and TGFβ, and further drive CSCC progression.

Article Snippet: Chromatin was immunoprecipitated using antibodies against WDR5 (13105, Cell Signaling Technology, USA), H3K4me3 (ab8580, Abcam, USA), and MLL1 (34907, Cell Signaling Technology, USA).

Techniques: Transformation Assay, Expressing

Journal: European Journal of Human Genetics

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: a Schematic of KMT2A protein structure (NP_001184033.1), with domains (and motifs) and the location of the identified de novo missense and splice variants. Domains were predicted by SMART program (http://smart.embl-heidelberg.de/). DNA binding AT hooks 1–3 (aa 169–180; aa 217–227; aa 301–309), CXXC domain (aa 1150–1198), zinc finger PHD-type 1–3 (aa 1431–1482; 1479–1533; 1566–1630), TAD domain (aa 2850–2858), bromodomain (aa 1703–1748), FYR N-terminal domain (aa 2021–2077), FYR-C terminal domain (aa 3666–3747), WDR5 interacting motif (aa 3765–3773), SET domain (aa 3832–3948), and post-SET domain (aa 3956–3972). b Sequence analysis of PCR products from the patient’s and parent’s (mother and father) DNA showing the different de novo variants. Asteriks indicate the position of the variants

Article Snippet: We used KMT2A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005933","term_id":"308199414","term_text":"NM_005933"}} NM_005933 ) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Binding Assay, Sequencing

Journal: European Journal of Human Genetics

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: Facial characteristics of the ID patients carrying a KMT2A variant. Patient 2 (P2) carries the c.8558T>G (p.(Met2853Arg)) variant, Patient 3 (P3) carries the c.3581G>A (p.(Cys1194Tyr)) variant and Patient 4 (P4) carries the c.11322–1G>A variant

Article Snippet: We used KMT2A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005933","term_id":"308199414","term_text":"NM_005933"}} NM_005933 ) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Variant Assay

Journal: European Journal of Human Genetics

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: a Sequence analysis of RT-PCR products from the patients (c.3460C>T (p.(Arg1154Trp)), c.8558T>G (p.(Met2853Arg)), c.11322–1G>A). In all cases, the two alleles were detected. b KMT2A mRNA expression in controls (WT) and mutated fibroblasts (c.3460C>T (p.(Arg1154Trp)), c.8558T>G (p.(Met2853Arg)), c.11322–1G>A). Y: KMT2A relative expression. Normalization factor was based on the GAPDH reference gene. Each chart represents one KMT2A relative quantification of a stimulated fibroblast culture. Errors bars represent SEM. c Western blot analysis of KMT2A protein of human control (WT1, WT2, WT3, and WT4; WT: all controls) and mutated fibroblasts (c.3460C>T (p.(Arg1154Trp)), c.8558T>G (p.(Met2853Arg)), c.11322–1G>A). GAPDH was used as loading control. Densitometry of western blotting was performed using Image J software. Data in arbitrary units (a.u.) represent the mean ± SEM of four separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 between control and KMT2A mutated fibroblasts. d Concerning the patient bearing the potential splice variant, RT-PCR using primers in exon 32 and 35 on cDNA from the patient and controls only showed one distinct band. Sequencing of the RT products showed two transcripts, the wild-type transcript and the mutated transcript with a deletion of the first nucleotide G of exon 33. This variant results in a premature stop-codon and is predicted to produce a truncated protein deleted of its C-terminal SET domain (p.Lys3775Serfs*32). This data suggests a change in the acceptor site of intron 32 which uses the mutated nucleotide A and the first G of the exon 33 to reconstitute the acceptor site

Article Snippet: We used KMT2A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005933","term_id":"308199414","term_text":"NM_005933"}} NM_005933 ) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Sequencing, Reverse Transcription Polymerase Chain Reaction, Expressing, Western Blot, Software, Variant Assay

Journal: European Journal of Human Genetics

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: Mapping nuclear targeting signals of wild-type (WT) and mutated KMT2A. Wild type or mutated KMT2A constructs (c.3460C>T (p.(Arg1154Trp)); c.8558T>G (p.(Met2853Arg))) were transiently transfected into COS7 cells and detected by staining with anti-MLL-1 (KMT2A) antibody. a Representative examples of typical patterns; uniform pattern and dot patterns (small dots or bigger patches absent within the nucleoli). b Distribution (% ± SEM) of the different nuclear patterns of cells expressing wild-type or mutated KMT2A constructs. Results were obtained by using data from more than 600 transfected cells of each construct in four independent experiments. The KMT2A c.3460C>T (p.(Arg1154Trp)) mutant abolishes significantly its capability to produce big dots (***χ 2 test with p < 0.0001)

Article Snippet: We used KMT2A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005933","term_id":"308199414","term_text":"NM_005933"}} NM_005933 ) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Construct, Transfection, Staining, Expressing, Mutagenesis

Journal: European Journal of Human Genetics

Article Title: Molecular and cellular issues of KMT2A variants involved in Wiedemann-Steiner syndrome

doi: 10.1038/s41431-017-0033-y

Figure Lengend Snippet: Expression of target genes of KMT2A (CDKN2C, CDKN1B, SIX2, HOXA9 and MEIS1) in wild-type fibroblasts (WT) and in human fibroblasts bearing different KMT2A variants (patients, c.3460C>T (p.(Arg1154Trp)); c.8558T>G (p.(Met2853Arg)), c.11322–1G>A).**p < 0.001, ***p < 0.0005 between control and KMT2A mutated fibroblasts

Article Snippet: We used KMT2A ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005933","term_id":"308199414","term_text":"NM_005933"}} NM_005933 ) Human ORF Clone (Origene, Rockville, USA) (pCMV6-AC-GFP backbone) for generate c.3460C>T (p.(Arg1154Trp)) and c.8558T>G (p.(Met2853Arg)) mutant using QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Les Ulis, France) and subcloning approaches.

Techniques: Expressing

Journal: Biomolecules & Therapeutics

Article Title: Isolation of MLL1 Inhibitory RNA Aptamers

doi: 10.4062/biomolther.2018.157

Figure Lengend Snippet: 3D-structure predictions for APT1-MLL1 interactions. (A) Predicted 3D-structure of APT1 was constructed. The numbers represent the order of nucleotide residues. Some nucleotide residues are indicated. (B) 3D-structure of the complex was calculated using NPDock web server and illustrated in two different views. MLL1 SET domain is represented as subdomains with different colors such as the N-flanking region in red, SET-N in dark yellow, SET-I in green, SET-C in cyan, and post-SET in blue. (C) Hydrogen bonds formed between APT1 and MLL1 protein in two regions (dotted lines). The bases interacting with MLL1 amino acids are illustrated.

Article Snippet: The MLL1 activity was measured using MLL1 Complex Chemiluminescent Assay Kit (BPS Bioscience, SD, USA) following the manufacturer’s protocol.

Techniques: Construct

Journal: Biomolecules & Therapeutics

Article Title: Isolation of MLL1 Inhibitory RNA Aptamers

doi: 10.4062/biomolther.2018.157

Figure Lengend Snippet: Evaluation of enrichment of MLL1-binding aptamers during SELEX. (A) Binding activity was measured using various amounts of S0, S3, and S16 libraries and 1 μg of MLL1 protein. The binding activity was calculated by qRT-PCR as described in “Materials and Methods”. Data are presented as means ± SEM. (B) Sequencing data of S0, S3, and S16 libraries were obtained using NGS. The percentage population of the five most popular abundant aptamers was revealed. (C) Sequence abundance in the sub-population consisting of the top 50 sequences. The percentage in the population of top 50 unique sequences was determined from S1, S3, and S16 libraries.

Article Snippet: The MLL1 activity was measured using MLL1 Complex Chemiluminescent Assay Kit (BPS Bioscience, SD, USA) following the manufacturer’s protocol.

Techniques: Binding Assay, Activity Assay, Quantitative RT-PCR, Sequencing

Journal: Biomolecules & Therapeutics

Article Title: Isolation of MLL1 Inhibitory RNA Aptamers

doi: 10.4062/biomolther.2018.157

Figure Lengend Snippet: Evaluation of binding affinity of aptamers. (A) 1 μg of APT1, APT2, APT3, and three libraries were incubated with 1 μg of MLL1 protein and the binding activity was determined by qRT-PCR analysis. The percentage of bound RNA was expressed. (B) Mutation or deletion was introduced in APT1 as illustrated. The randomized region of APT1 are enclosed in a box, mutated nucleotides are underlined, and the deleted nucleotides are marked with hyphen. (C) The binding activity of each variant (1 μg) was measured by qRT-PCR analysis and the amount of bound RNA was calculated. Relative amount of bound RNAs was expressed as a fold change using wild-type APT1 was used as a control.

Article Snippet: The MLL1 activity was measured using MLL1 Complex Chemiluminescent Assay Kit (BPS Bioscience, SD, USA) following the manufacturer’s protocol.

Techniques: Binding Assay, Incubation, Activity Assay, Quantitative RT-PCR, Mutagenesis, Variant Assay

Journal: Biomolecules & Therapeutics

Article Title: Isolation of MLL1 Inhibitory RNA Aptamers

doi: 10.4062/biomolther.2018.157

Figure Lengend Snippet: Effect of aptamers on MLL1 activity. MLL1 activity was measured using MLL1 Complex Chemiluminescent Assay Kit. S-adenosylmethionine was added as a methyl-group donor. MLL1 (2 ng/μl) was incubated with or without aptamers. S0 library was used as a control. * p <0.05, *** p <0.005 versus control. Data are presented as means ± SEM. Statistical significance was determined with unpaired Student’s t -test.

Article Snippet: The MLL1 activity was measured using MLL1 Complex Chemiluminescent Assay Kit (BPS Bioscience, SD, USA) following the manufacturer’s protocol.

Techniques: Activity Assay, Incubation

Journal: Cell Death & Disease

Article Title: Alcohol-abuse drug disulfiram targets pediatric glioma via MLL degradation

doi: 10.1038/s41419-021-04078-9

Figure Lengend Snippet: A Array data analysis derived from Bax et al. . In the heat map, the relative expression levels of MLL1 and MLL2 are shown. B SF188 cells were seeded in six-well plates at a density of 0.4 × 10 6 cells/well and transfected with 25 pmol ON-TARGETplus Control pool Non-Targeting pool (labeled C), or ON-TARGETplus SMART pool siRNA targeting MLL1 and MLL2 (labeled siRNA). Cell viability assays were performed on the indicated days. Data are mean ± STD; n = 3; ∗ p < 0.05, ∗∗ p < 0.01; student t -test. C GSEA showing significant negative enrichment of the WANG_MLL_TARGETS gene set after MLL1/MLL2 downregulation in SF188 cells. The gene set comprises genes that require MLL1 for H3K4me3 and expression in mouse embryonic fibroblast cells. D GSEA showing significant negative enrichment of the MLL2_TARGETS gene set for MLL1/MLL2 downregulation in SF188. The gene set comprises previously described MLL2 targets (see material and method). E GSEA showing significant negative enrichment of genes that mediate apoptosis by activation of caspases (MSigDB gene set: HALLMARK_APOPTOSIS) for MLL1/MLL2 downregulation in SF188 cells. F GSEA showing significant negative enrichment of Hox cluster genes for MLL1/MLL2 downregulation in SF188 cells. The gene set comprises all Hox cluster genes. The heatmap represents Hox cluster genes expression in the three independent experiments. Genes present in the core enrichment signature are highlighted in light green, genes marked with an asterisk were further analyzed by qPCR. G : qPCR analyses of selected HOX genes expression. Data are mean ± STD; n = 3; ∗ p < 0.05, ∗∗ p < 0.01; one-sample t -test. H GSEA showing significant negative enrichment of transcription factors with aberrant expression in glioblastoma as reported by Rheinbay et al. . I and J qPCR analyses of selected transcription factors expression upon MLL1/MLL2 downregulation. Data are mean ± STD; n = 3; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-sample t -test. k GSEA showing downregulation of MYC targets genes set for MLL1/MLL2 downregulation in SF188 cells.

Article Snippet: Two micrograms of MLL1 (Bethyl, A700-010) or H3K4me2 (Abcam, ab32356) antibodies were used.

Techniques: Derivative Assay, Expressing, Transfection, Control, Labeling, Activation Assay

Journal: Cell Death & Disease

Article Title: Alcohol-abuse drug disulfiram targets pediatric glioma via MLL degradation

doi: 10.1038/s41419-021-04078-9

Figure Lengend Snippet: A – E Top: whole-cell lysates were prepared from the indicated cell lines, cultured in the absence or presence of 0.5 mM auranofin with increasing concentrations of disulfiram for 16 h. Extracts were probed with α-MLL1 antibody and α-vinculin antibody as loading control. Bottom: the graphs represent image analysis of band intensity. Data are mean ± STD; n = 3; ∗ p < 0.05, ∗∗ p < 0.01 ∗∗∗ p < 0.001; two-way analysis of variance (ANOVA) test with Bonferroni posttest.

Article Snippet: Two micrograms of MLL1 (Bethyl, A700-010) or H3K4me2 (Abcam, ab32356) antibodies were used.

Techniques: Cell Culture, Control

Journal: Cell Death & Disease

Article Title: Alcohol-abuse drug disulfiram targets pediatric glioma via MLL degradation

doi: 10.1038/s41419-021-04078-9

Figure Lengend Snippet: A Cells were plated in 96-wells plates. The following day cells were treated with the indicated concentrations of disulfiram in the absence or presence of increasing doses of auranofin. At the indicated time cell viability assays were performed. Data are mean ± STD; n = 3. B Top: whole-cell lysates were prepared from the indicated cells, cultured in the absence or presence of increasing concentrations of disulfiram for 16 h. Extracts were probed with α-MLL1 antibody and α-vinculin antibody as loading control. Bottom: the graphs represent image analysis of band intensity. Data are mean ± STD; n = 3; ∗ p < 0.05; two-way analysis of variance (ANOVA) test with Bonferroni posttest. C GPC16 cells were seeded in six-well plates at a density of 0.4 × 10 6 cells/well and transfected with 25 pmol ON-TARGETplus Control pool Non-Targeting pool (labeled C), or ON-TARGETplus SMART pool siRNA targeting MLL1 and MLL2 (labeled siRNA). Cell viability assays were performed 3 days after transfection. Data are mean ± STD; n = 3; ∗ p < 0.05; one-sample t -test. D , E , and F same experiment as Fig. but with GPC16 cells. G qPCR analyses of selected transcription factors expression upon MLL1/MLL2 downregulation in GPC16 cells. Data are mean ± STD; n = 3; ∗∗ p < 0.01; one-sample t -test.

Article Snippet: Two micrograms of MLL1 (Bethyl, A700-010) or H3K4me2 (Abcam, ab32356) antibodies were used.

Techniques: Cell Culture, Control, Transfection, Labeling, Expressing

Journal: Cell Death & Disease

Article Title: Alcohol-abuse drug disulfiram targets pediatric glioma via MLL degradation

doi: 10.1038/s41419-021-04078-9

Figure Lengend Snippet: A and B GSEA showing downregulation of MLL1/MLL2 core genes set (genes downregulated upon MLL1/MLL2 downregulation) in disulfiram-treated SF188 and GPC16 cells. C GSEA showing downregulation of HOX cluster genes set (gene set including all 39 human Hox cluster genes) in disulfiram-treated SF188 cells. The heatmap is part of the GSEA. It represents Hox cluster genes expression in disulfiram-treated (DSF) and untreated cells (CTRL) with three replicates each. Genes present in the core enrichment signature are highlighted in green, genes marked with an asterisk were further analyzed. C qPCR analyses of HOX genes expression in disulfiram-treated SF188 cells. Data are mean ± STD; n = 3; ∗ p < 0.05, ∗∗ p < 0.01; one-sample t -test. D qPCR analyses of transcription factors expression upon disulfiram treatment. Data are mean ± STD; n = 3; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-sample t -test. E Western blot analysis of MYC and PAX2 expression level upon disulfiram treatment. Data are mean ± STD; n = 3; ∗ p < 0.05, t -test. F GSEA showing downregulation of MYC targets genes set (genes upregulated upon MYC expression) in disulfiram-treated SF188 cells (MSigDB gene set: SCHUHMACHER_MYC_TARGETS_UP). G GSEA showing downregulation of aberrant transcription factor set (75 transcription factors with aberrant expression in glioblastoma as reported by Rheinbay et al.) in disulfiram-treated GPC16 cells. H qPCR analyses of OLIG2 , SOX2 , and MYC expression in disulfiram-treated GPC16 cells. Data are mean ± STD; n = 3; ∗∗ p < 0.01; one-sample t -test. I Western blot analysis of SOX2 and MYC expression level upon disulfiram treatment. Data are mean ± STD; n = 3; ∗ p < 0.05, t -test.

Article Snippet: Two micrograms of MLL1 (Bethyl, A700-010) or H3K4me2 (Abcam, ab32356) antibodies were used.

Techniques: Expressing, Western Blot

Journal: Oncotarget

Article Title: HoxA9 regulated Bcl-2 expression mediates survival of myeloid progenitors and the severity of HoxA9-dependent leukemia

doi:

Figure Lengend Snippet: (A) Expression of HoxA9 and Bcl-2 in human leukemic lines. Total cell lysates were harvested and probed with antibodies against HoxA9, Bcl-2 and actin was used as a loading control. * Unknown band, which may be a HoxA9 splice variant. (B) Sensitivity of human leukemic lines expressing HoxA9 to Bcl-2 inhibitor ABT-199. Human leukemic cells expressing higher levels of endogenous HoxA9 were treated or not with 1μM of ABT199 for 48hs. Cell survival was determined by flow cytometric analysis of PI exclusion. Results show means ±SEM of 3 independent experiments. (C) Expression of HoxA9 (2 probesets – 209905_at and 214651_s_at) and Bcl-2 (one probeset – 203685_at) from 285 AML samples grouped into each of 16 clusters characterized by gene expression profiles previously defined by Valk et al (GSE1159). Where a cluster is also associated with a particular genetic lesion, this is shown on the right of each cluster and where no such association exists this is shown as “none”. Bright bars indicate the clusters characterized by significant association between HoxA9 and Bcl-2 expression. Expression for each cluster is shown relative to the normal control group (NBM and CD34+, n=8). Significant genes were defined as BH-adjusted p<0.05. (D) Expression of MLL-AF9, HoxA9 and Bcl-2 in MLL-AF9 immortalized cell lines. Hematopoietic progenitor cells were immortalized by infection with lentivirus encoding MLL-AF9 under the control of a tetracycline-repressible promoter. Cells were cultured in the absence (Day 0) or presence of 100ng/ml doxycycline for 3 and 5 days. Lysates were harvested and probed with antibodies against MLL, HoxA9, Bcl-2 and actin was used as a loading control.

Article Snippet: The antibodies used were; Anti-mouse Bcl-2 mAb (BD Biosciences), anti-Actin mAb and pAb anti-Bax and anti-Bak (Sigma), anti-Bad pAb (Cell Signaling), anti-Bim pAb (Stressgen), anti-Puma pAb (ProSci), anti-HoxA9 pAb and anti-MLL clone N4.4 mAb (Millipore), anti-Mcl-1 pAb (Rockland) and anti-Bcl-x L pAb (R&D Systems). pAb anti-Bid, anti-Bmf and anti-A1 pAb were a kind gift from Andreas Strasser, Lorraine O'Reilly and Marco Herold respectively.

Techniques: Expressing, Variant Assay, Infection, Cell Culture

Journal: Oncotarget

Article Title: HoxA9 regulated Bcl-2 expression mediates survival of myeloid progenitors and the severity of HoxA9-dependent leukemia

doi:

Figure Lengend Snippet: (A-B) Survival of AML-burdened mice after transplantation with HoxA9/Meis1- (left panel) or MLL-AF9- (right panel) transformed cells of wild-type (filled lines) and Bcl-2 −/− (dashed lines)genotypes. Number of mice used in each group indicated in brackets (wt/ Bcl-2 −/− ). GFP control mice (dotted lines) from the same experiment did not develop disease independent of the genotype, thus confirming the potential of engraftment of Bcl-2 −/− . (***) P < 0.0005; (****) P <0.0001. All mice transplanted with Bcl-2 −/− transformed AML cells presented a low white blood cell count compared to wild-type transplanted mice. Shown are the total numbers of white blood cells in peripheral blood ±SEM of mice bearing HoxA9/Meis1 (C) or MLL-AF9 (D) induced AML of the indicated genotype; (E and F) Representative data of the proportion of cells with morphologic features of blasts following May Grunwald Giemsa staining of blood smears preparation. Magnification 40x or inset 100x. Scale bar 100μm.

Article Snippet: The antibodies used were; Anti-mouse Bcl-2 mAb (BD Biosciences), anti-Actin mAb and pAb anti-Bax and anti-Bak (Sigma), anti-Bad pAb (Cell Signaling), anti-Bim pAb (Stressgen), anti-Puma pAb (ProSci), anti-HoxA9 pAb and anti-MLL clone N4.4 mAb (Millipore), anti-Mcl-1 pAb (Rockland) and anti-Bcl-x L pAb (R&D Systems). pAb anti-Bid, anti-Bmf and anti-A1 pAb were a kind gift from Andreas Strasser, Lorraine O'Reilly and Marco Herold respectively.

Techniques: Transplantation Assay, Transformation Assay, Cell Counting, Staining