Journal: Haematologica

Article Title: Impact of Sox11 over-expression in Ba/F3 cells

doi: 10.3324/haematol.2018.197467

Figure Lengend Snippet: Phenotypical, proliferative and transcriptional changes in Ba/F3 cells upon 72 h of induced Sox11 expression. (A) Western Blot of SOX11 protein expression in Sox11-ON (doxycycline supplemented medium) and Sox11-OFF (control medium) cells at 72 h, detected with the rabbit polyclonal anti-SOX11 antibody HPA000536, Sigma-Aldrich. B) Bright field microscopy images of cell aggregates following 72 h of continuous Sox11 expression (10x), imaged by Nikon Ti-E microscope. C) Sox11-ON cells incorporates less 3H-Thymidine at 72 h of Sox11 induction following a 4 h pulse, as compared to Sox11-OFF cells, measured in counts per minute, error bars represent the standard deviation (P=0.0086, n=3). D) Mean relative absorption measured by XTT (n=3). Metabolic activity is significantly reduced (P=0.0124) in Sox11-ON cells as compared to Sox11-OFF cells. E) Volcano plot representation of transcript level differences by Affymetrix MTA-1 mouse arrays (Microarray data has been made available through the GEO database with accession number GSE108419). Names are shown for the genes with the largest transcript level fold change (log2FC ≥1.3 or ≤−1.3). Blue and red: genes with significantly altered transcript levels in Sox11-ON cells and a fold change below -2 (blue) (FDR q-value ≤0.05 and log2FC ≥−1) or above 2 (red) (FDR q-value ≤0.05 and log2FC ≥1); gray: genes significantly changed at the transcript level (FDR q-value ≤0.05); black: genes with non-significant transcript level changes. F) Expression levels after Sox11 induction in genes specifically expressed at different stages of B-cell development. Only the pro-B restricted genes Id1 and Tal1 had significantly altered transcript levels in Sox11-ON cells (FDR q-value: 0.006 and 0.016, respectively). None of the other investigated pro-B and pre-B cell associated genes were altered at the transcript level. Genes associated with later B-cell developmental stages are shown for comparison. Transcript levels are presented as a gene-wise standardized expression (Z-score). FC: fold change.

Article Snippet: E) Volcano plot representation of transcript level differences by Affymetrix MTA-1 mouse arrays (Microarray data has been made available through the GEO database with accession number GSE108419).

Techniques: Expressing, Western Blot, Microscopy, Standard Deviation, Activity Assay, Microarray

Journal: Cancer research

Article Title: The role of metastasis-associated protein 1 in prostate cancer progression.

doi: 10.1158/0008-5472.can-03-2755

Figure Lengend Snippet: Fig. 1. A, heatmap of the cDNA microarray data for benign prostatic tissue (Benign), localized pros- tate cancer (PCA), and metastatic prostate cancer (METS) samples [modified from Varambally et al. (2)]. Metastasis-associated protein 1 (MTA1) was among several genes up-regulated in METS when compared with PCA or benign prostate tissue. B, error bars with 95% confidence intervals (CIs) demonstrating the expression level of MTA1 in benign prostatic tissue, PCA, and METS cases. The level of MTA1 expression in PCA was signifi- cantly increased compared with benign prostate tissue. METS showed the highest level of MTA1 expression. C, Western blot analysis demonstrating MTA1 protein expression. Fifteen g of protein extract of normal, PCA, and METS tissue was separated in a 10% polyacrylamide gel. Incubation with anti-MTA1 antibody 1:50 overnight at 4°C. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to demonstrate equal protein loading. The bar graph shows normalized expres- sion as MTA1/GAPDH ratio. Normal samples showed only little MTA1 expression, whereas the majority of localized prostate cancer and almost all of the metastatic prostate cancer samples have a strong increase in MTA1 expression. D, represent- ative images of tissue microarray samples staining absent, weak, moderate, and strong for MTA1 ex- pression. MTA1 expression was confined to the nuclei of cells as demonstrated by immunohisto- chemistry (200 magnification). E, error bars with 95% CIs demonstrating MTA1 protein expres- sion levels in a wide spectrum of prostate tissue samples. MTA1 staining intensity was evaluated along a scale ranging from negative expression (score 1) to strong staining (score 4). F, Kaplan-Meier analyses of MTA1 status and pros- tate-specific antigen (PSA)-defined recurrence-free survival after radical prostatectomy for clinically localized prostate cancer. Biochemical failure was defined as a PSA elevation 0.2 ng/ml after rad- ical prostatectomy. Negative MTA1 expression was associated with a 2.8 relative risk (95% CI: 1.2–6.7, P 0.02) of PSA recurrence as compared with moderate/strong MTA1 expression.

Article Snippet: The primary antibody against MTA1 [anti-MTA1 monoclonal (A11), sc-17773; Santa Cruz Biotechnology] was incubated overnight in a 1:5 dilution at 4°C.

Techniques: Microarray, Modification, Expressing, Western Blot, Incubation, Staining, Immunohistochemistry

Journal: Experimental hematology

Article Title: RNAi Screen Identifies MTA1 as an Epigenetic Modifier of Differentiation Commitment in Human HSPCs.

doi: 10.1016/j.exphem.2022.08.004

Figure Lengend Snippet: Figure 1 Pooled short hairpin (sh)RNA screen identifies MTA1 as a regulator of human hematopoietic stem and progenitor cell (HSPC) propagation in vitro. (A) Overview of the screen. Primary cord blood-derived CD34+ cells were transduced with pools of a len- tiviral library containing 45,000 shRNAs. CD34+ cells were magnetically isolated after 20 days of culture to determine changes in shRNA distribution between days 3 and 20. (B) Genes ranked based on the median shRNA log2 fold change (log2 FC) between days 20 and 3. (C) Distribution of the MTA1-targeting shRNAs in the library. (D) Relative mRNA and protein levels of MTA1 in cord blood (CB)-derived CD34+ cells after knockdown by shRNAs. (E) Representative flow cytometry plots of CD34 and CD90 staining of GFP+

Article Snippet: Chromatin immunoprecipitation (ChIP)-grade Protein A/G Magnetic Beads (Fisher Scientific) were incubated with MTA1 antibody (Cell Signaling Technology, Danvers, MA USA) for 30 min or rabbit monoclonal antibody (Cell Signaling Technology) as an isotype control.

Techniques: In Vitro, Derivative Assay, Transduction, Isolation, shRNA, Knockdown, Cytometry, Staining

Journal: Experimental hematology

Article Title: RNAi Screen Identifies MTA1 as an Epigenetic Modifier of Differentiation Commitment in Human HSPCs.

doi: 10.1016/j.exphem.2022.08.004

Figure Lengend Snippet: Figure 2 Knockdown of MTA1 preserves the immature phenotype of hematopoietic stem and progenitor cells (HSPCs) and perturbs differentiation into myeloid and erythroid lineages. (A) Representative flow cytometry plots of CD34, CD90, and EPCR staining of short hairpin (sh)RNA-transduced CD34+CD90+CD38−CD45RA−cells following 1 week of culture. (B) Relative fold increase in

Article Snippet: Chromatin immunoprecipitation (ChIP)-grade Protein A/G Magnetic Beads (Fisher Scientific) were incubated with MTA1 antibody (Cell Signaling Technology, Danvers, MA USA) for 30 min or rabbit monoclonal antibody (Cell Signaling Technology) as an isotype control.

Techniques: Knockdown, Cytometry, Staining

Journal: Experimental hematology

Article Title: RNAi Screen Identifies MTA1 as an Epigenetic Modifier of Differentiation Commitment in Human HSPCs.

doi: 10.1016/j.exphem.2022.08.004

Figure Lengend Snippet: Figure 3 Knockdown of MTA1 impairs in vivo reconstitution of transplanted hematopoietic stem and progenitor cells (HSPCs). (A) Engraftment of human CD34+ cells transduced with MTA1 or Scr short hairpin (sh)RNAs and transplanted without prior sorting to sublethally irradiated NSG (NOD Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice (five mice for each condition). The graph indicates the frequency of total hCD45+ and hCD45+GFP+ cells in peripheral blood. (B) Engraftment of human GFP+CD34+CD38−CD90+CD45RA−cells transduced with MTA1 or Scr shRNAs sorted for Green Fluorescent Protein (GFP) expression before transplantation to sublethally irradiated NSG mice. The graphs indicate the percentages of hCD45+GFP+ cells in peripheral blood and bone marrow after 16 weeks (n = 12).

Article Snippet: Chromatin immunoprecipitation (ChIP)-grade Protein A/G Magnetic Beads (Fisher Scientific) were incubated with MTA1 antibody (Cell Signaling Technology, Danvers, MA USA) for 30 min or rabbit monoclonal antibody (Cell Signaling Technology) as an isotype control.

Techniques: Knockdown, In Vivo, Transduction, Irradiation, Expressing, Transplantation Assay

Journal: Experimental hematology

Article Title: RNAi Screen Identifies MTA1 as an Epigenetic Modifier of Differentiation Commitment in Human HSPCs.

doi: 10.1016/j.exphem.2022.08.004

Figure Lengend Snippet: Figure 4 MTA1 associates with members of the nucleosome remodeling deacetylase (NuRD) complex in hematopoietic stem and progenitor cells (HSPCs) and influences H3K27 deacetylation. (A) Composition of the NuRD complex and its enzymatic activities. (B) Volcano plot of MTA1 interactome in CD34+ cell lysates. Proteins highlighted in red are NuRD complex members, which were immu- noprecipitated and identified to interact with MTA1. (C) Global H3K27Ac levels measured by Western blot in cord blood (CB)-derived CD34+ cells following MTA1 KD by short hairpin (sh)RNAs. (D) Schematic overview of the microarray experiment. CD34+CD38−CD90+CD45RA−cells were transduced with shRNA vectors, and GFP+ cells were sorted 60 hours after transduction for analysis. (E) Volcano plot of up- or downregulated genes plotted by fold change (x axis) and p value (y axis) between Scr control and MTA1 shRNA transduced cells. More than 1.5 times significantly (p < 0.05) up- or downregulated genes are highlighted in green and red, respectively. (F) Commonly up- and downregulated genes between sh1 and sh2 shRNAs targeting MTA1 compared with Scr control. (G) Heatmap of the expression of erythroid lineage-specific genes, which were downregulated in MTA1 KD cells.

Article Snippet: Chromatin immunoprecipitation (ChIP)-grade Protein A/G Magnetic Beads (Fisher Scientific) were incubated with MTA1 antibody (Cell Signaling Technology, Danvers, MA USA) for 30 min or rabbit monoclonal antibody (Cell Signaling Technology) as an isotype control.

Techniques: Histone Deacetylase Assay, Western Blot, Derivative Assay, Microarray, Transduction, shRNA, Control, Expressing

Journal: Journal of Biological Chemistry

Article Title: MTA1 Coregulation of Transglutaminase 2 Expression and Function during Inflammatory Response

doi: 10.1074/jbc.m110.199273

Figure Lengend Snippet: FIGURE 1. MTA1 regulation of TG2 expression. A, TG2 mRNA expression in wild-type and MTA1/ MEFs identified by using microarray gene expres- sion profiling. B, qPCR analysis of TG2 mRNA expression in wild-type and MTA1/ MEFs. Results represent a -fold change decrease in TG2 mRNA expression in MTA1/ MEFs relative to the wild type. Each value repre- sents the means S.E. of three independent experiments. C, Western blot analysis of TG2 protein expression from the cell lysates isolated from wild- type, MTA1/, and MTA1/ MEFs overexpressing MTA1. D, RT-PCR analy- sis of TG2 expression in HC11 cells stably expressing pcDNA and MTA1. RT- PCR analysis was carried out with the cDNA synthesized from the 2 g of total RNA isolated from the HC11/pcDNA and HC11/MTA1 stable cells using the gene-specific primers. E, qPCR analysis of TG2 and MTA1 mRNA expres- sion in MCF10A, MCAF10AT1, MCF10CA1D, and MCF10DCIS cells. qPCR was carried out for the TG2, MTA1, and -actin mRNAs from the RNA isolated from the above mentioned cell lines, and results were presented in terms of -fold change after normalizing with -actin mRNA levels. 2 g of total RNA from each cell line was used for cDNA synthesis. Each value represents the means S.E. of three independent experiments. F, qPCR analysis of TG2 expression in MCF-7 cells after selective knockdown of MTA1 using MTA1 siRNA. Results presented were the means S.E. of three independent ex- periments. The upper panel represents the Western blot analysis showing the effective knockdown of MTA1 by using MTA1-specific siRNA. G, Western blot analysis for MTA1 knockdown using MTA1 siRNA in the MCF-7 cells used in panel H. H, confocal analysis representing the TG2 protein expres- sion in MCF-7 cells after selective knockdown of MTA1 using MTA1 siRNA.

Article Snippet: Antibodies against MTA1 (catalogue number A300-280A) and RNA polymerase II (pol II) (catalogue number A300-653A) were purchased from Bethyl Laboratories, whereas normal mouse IgG, rabbit IgG, and antibodies against vinculin were from Sigma.

Techniques: Expressing, Microarray, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Stable Transfection, Synthesized, cDNA Synthesis, Knockdown

Journal: Journal of Biological Chemistry

Article Title: MTA1 Coregulation of Transglutaminase 2 Expression and Function during Inflammatory Response

doi: 10.1074/jbc.m110.199273

Figure Lengend Snippet: FIGURE 2. Induction of TG2 expression by LPS is MTA1-mediated. A, RT- PCR analysis of TG2 and MTA1 expression in the cDNA synthesized from 2 g of the total RNA isolated from Raw cells after treating with LPS (1 g/ml of the medium) for 2 and 4 h. B, RT-PCR analysis of TG2 and MTA1 expres- sion in LPS-treated Raw cells (1 g/ml of the medium) with or without MTA1 knockdown. After 24 h of MTA1 siRNA treatment, Raw cells were stimulated with LPS (1 g/ml of the medium) for 4 h, and RNA was isolated for RT-PCR analysis. C, qPCR analysis of TG2 expression in macrophages iso- lated from MTA1/ and MTA1/ mice. Results represent the decreased -fold change in the TG2 mRNA expression after normalizing with -actin mRNA levels in macrophages isolated from MTA1/ mice after treating with LPS for 2 h. Results presented were the means S.E. of three inde- pendent experiments. D, Western blot analysis for TG2 expression from the lysates of wild-type and MTA1/ MEFs treated with LPS (1 g/ml medium) at time intervals of 2, 4, and 8 h.

Article Snippet: Antibodies against MTA1 (catalogue number A300-280A) and RNA polymerase II (pol II) (catalogue number A300-653A) were purchased from Bethyl Laboratories, whereas normal mouse IgG, rabbit IgG, and antibodies against vinculin were from Sigma.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Synthesized, Isolation, Knockdown, Western Blot

Journal: Journal of Biological Chemistry

Article Title: MTA1 Coregulation of Transglutaminase 2 Expression and Function during Inflammatory Response

doi: 10.1074/jbc.m110.199273

Figure Lengend Snippet: FIGURE 3. MTA1 regulates TG2 transcription. A, TG2 promoter-Luc activity in LPS- (1 g/ml medium for 4 h) stimulated Raw cells with or without MTA1 knockdown. After MTA1 knockdown using MTA1 siRNA, Raw cells were transfected with murine TG2-pGL3 luciferase reporter plasmid and pCMV vector carrying the -galactosidase gene as an internal control for transfec- tion efficiency. After 36 h of transfection, cells were treated with LPS (1 g/ml of the medium for 4 h), and luciferase activities in triplicate samples were measured. Results were presented in terms of -fold change, and the values represent the means S.E. from three independent transfection experiments. B, TG2 promoter-Luc activity in LPS- (1 g/ml of the medium for 4 h) stimulated wild-type and MTA1/ MEFs. The TG2-pGL3 luciferase assay was carried out as described for panel A. Results were presented in terms of -fold change, and the values represent the means S.E. from three independent transfection experiments. C, Raw cells were transfected with the indicated plasmids, and after 36 h of transfection, cells were treated with LPS (1 g/ml of the medium) for 4 h. Luciferase activity was calculated as mentioned earlier. The upper panel represents the Western blot analysis for T7-MTA1 to show the transfection efficiency.

Article Snippet: Antibodies against MTA1 (catalogue number A300-280A) and RNA polymerase II (pol II) (catalogue number A300-653A) were purchased from Bethyl Laboratories, whereas normal mouse IgG, rabbit IgG, and antibodies against vinculin were from Sigma.

Techniques: Activity Assay, Knockdown, Transfection, Luciferase, Plasmid Preparation, Control, Western Blot

Journal: Journal of Biological Chemistry

Article Title: MTA1 Coregulation of Transglutaminase 2 Expression and Function during Inflammatory Response

doi: 10.1074/jbc.m110.199273

Figure Lengend Snippet: FIGURE 4. TG2 is an MTA1 target gene. A, line diagram showing the binding of a region of MTA1 on murine TG2 promoter. To carry out ChIP-based pro- moter walk, murine TG2 promoter was divided into five regions. The double-headed arrow represents the MTA1 binding region on murine TG2 promoter. B, recruitment of MTA1 to TG2 chromatin in Raw cells treated with or without LPS for 1 h. Raw cells that were treated with 1% formaldehyde to cross-link the histones to DNA were lysed by sonication and immunoprecipitated by either anti-MTA1 antibody or IgG antibody. The immunoprecipitates (IP) were collected by adding beads; beads were washed, DNA was eluted from the beads, and purified DNA was subjected to PCR. C, double ChIP analysis of recruit- ment of MTA1-pol II complex onto the TG2-chromatin (630 to 849) in Raw cells treated with LPS for 1 h. The first ChIP was carried out with anti-MTA1 antibody followed by second ChIP with anti-RNA polymerase II. From the same elutes of ChIP analysis, qPCR analysis was also performed.

Article Snippet: Antibodies against MTA1 (catalogue number A300-280A) and RNA polymerase II (pol II) (catalogue number A300-653A) were purchased from Bethyl Laboratories, whereas normal mouse IgG, rabbit IgG, and antibodies against vinculin were from Sigma.

Techniques: Binding Assay, Sonication, Immunoprecipitation, Purification, ChIP-qPCR

Journal: Journal of Biological Chemistry

Article Title: MTA1 Coregulation of Transglutaminase 2 Expression and Function during Inflammatory Response

doi: 10.1074/jbc.m110.199273

Figure Lengend Snippet: FIGURE 6. TG2 is an NF-B target gene. A, ChIP analysis of p65RelA fol- lowed by MTA1 recruitment to TG2-chromatin in Raw cells p65RelA, treated with LPS for 1 h. ChIP analysis was carried with either p65RelA antibody or double ChIP analysis; the first ChIP was with p65RelA antibody, and the sec- ond ChIP was with MTA1 antibody from the lysates of LPS-stimulated Raw cells. B, recruitment of p65 followed by pol II on to TG2 chromatin (630 to 849). The lower panel represents qPCR-ChIP. IP, immunoprecipitates. C, ef- fect of parthenolide (5 M) on the recruitment of p65 onto TG2 promoter (630 to 849) in LPS- (1 g/ml of the medium for 1 h) stimulated Raw cells. Raw cells treated with parthenolide 1 h prior to LPS treatment were used to carry out ChIP analysis as described in the legend for Fig. 4. The lower panel represents the qPCR-ChIP. D, Western blot analysis for MTA1 and NF-B-p65 after immu- noprecipitating with MTA1 in Raw cells treated with LPS. E, in vitro interaction of MTA1 and p65. The GST-p65 fusion protein and GST were used in a GST pull- down assay with in vitro-translated 35S-labeled full-length MTA1 (MTA1 (S35)). F, EMSA analysis of p65 and MTA1 binding to the mouse TG2 promoter using the wild-type and mutant oligonucleotides encompassing NF-B consensus sequences in Raw cells treated with LPS for 1 h. G, summary of ChIP analysis carried out on the mouse TG2 promoter. This figure summarizes the recruit- ment of MTA1, p65RelA, MTA1-pol II complex, and p65-MTA1 complex onto the TG2 chromatin region (630 to 849).

Article Snippet: Antibodies against MTA1 (catalogue number A300-280A) and RNA polymerase II (pol II) (catalogue number A300-653A) were purchased from Bethyl Laboratories, whereas normal mouse IgG, rabbit IgG, and antibodies against vinculin were from Sigma.

Techniques: Western Blot, In Vitro, Pull Down Assay, Labeling, Binding Assay, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: MTA1 Coregulation of Transglutaminase 2 Expression and Function during Inflammatory Response

doi: 10.1074/jbc.m110.199273

Figure Lengend Snippet: FIGURE 7. Schematic representation of MTA1 regulation of TG2 and its impact on LPS response. LPS stimulation of peritoneal macrophages re- sults in the induction of MTA1 and NF-B. Once MTA1 is induced, it acts as a coactivator and gets recruited to the TG2 chromatin along with p65RelA, leading to increased TG2 expression, resulting in constitutive activation of NF-B, triggering activation of the inflammation process.

Article Snippet: Antibodies against MTA1 (catalogue number A300-280A) and RNA polymerase II (pol II) (catalogue number A300-653A) were purchased from Bethyl Laboratories, whereas normal mouse IgG, rabbit IgG, and antibodies against vinculin were from Sigma.

Techniques: Expressing, Activation Assay