Journal: Oncotarget

Article Title: An AML1-ETO/miR-29b-1 regulatory circuit modulates phenotypic properties of acute myeloid leukemia cells

doi: 10.18632/oncotarget.18127

Figure Lengend Snippet: A. Top schematic shows the miR-29a/b-1 locus on human chromosome 7 (Accession Number EU154353) . Runx binding sites are indicated. RNAs originating from the locus are shown; orange blocks indicate exons, while the orange line shows intronic regions. Genomic tracks from ChIP-seq dataset from Trombly et al were re-analyzed for occupancy of the miR-29a/b-1 locus by AML1-ETO, NCoR, or the activating H3K4me3 and suppressive H3K27me3 histone modifications. B. Actively proliferating Kasumi-1 cells were subjected to chromatin immunoprecipitation with two separate antibodies against ETO (black bars), a rabbit polyclonal antibody against carboxy-terminus of RUNX1, which is not present in AML1-ETO (white bar), and an IgG isotype control (gray bar). Immuno-enriched chromatin was analyzed by quantitative PCR using primers encompassing Runx binding sites in the regulatory region of the miR-29a/b-1 locus. Bar graph shows immuno-enriched chromatin as a percentage of input. C . Western blot analysis of SKNO-1 cells infected with a non-silencing short hairpin RNA (shNS), or an shRNA selectively targeting AML1-ETO (shA/E). Significant knock down of the AML1-ETO protein was reproducibly obtained 5 days post-infection. GAPDH was used as protein loading control. D. Total cellular RNA from SKNO-1 cells infected with shNS and shA/E was analyzed for expression of the pri-miR-29a/b-1, as well as of CDKN1A , a known AML1-ETO target gene. Bar graph represents an average of three independent experiments, and error bars indicate standard deviation (SD). Student's t-test shows highly significant changes in the expression of pri-miR-29b-1 and CDKN1A ( p > 0.01) upon AML1-ETO knockdown.

Article Snippet: Following antibodies were used in this study: RUNX1 (Cat. No. 4334, Cell Signaling Technology, Inc., Danvers, MA), ETO (Cat. No. 4498S, Cell Signaling Technology, Inc.), RUNX1T1/ETO (Cat. No. ab124269; Abcam, Cambridge, MA), CDK6 Clone DCS83 (Cat. No. 3136S; Cell Signaling Technology, Inc.), Tubulin Clone DM1A (Cat. No. T9026; Sigma-Aldrich, St. Louis, MO), and normal (Cat. No. sc-2027 for anti-rabbit; Cat. No. sc-2025 for anti-mouse) and HRP-conjugated IgG (Cat. No. sc-2004 for anti-rabbit; Cat. No. sc-2005 for anti-mouse) antibodies (Santa Cruz Biotechnology, Dallas, TX).

Techniques: Binding Assay, ChIP-sequencing, Chromatin Immunoprecipitation, Control, Real-time Polymerase Chain Reaction, Western Blot, Infection, shRNA, Knockdown, Expressing, Standard Deviation

Journal: Oncotarget

Article Title: An AML1-ETO/miR-29b-1 regulatory circuit modulates phenotypic properties of acute myeloid leukemia cells

doi: 10.18632/oncotarget.18127

Figure Lengend Snippet: A. The ETO/RUNX1T1 transcripts have seed sequences in 3’UTRs for conserved miR families that can potentially down-regulate the proteins. The seed sequence for the miR-29 family is shown. B. 293T cells were co-transfected with a ETO 3’UTR-luciferase reporter construct and NS, miR-15 or miR-29 mimics. Cells were harvested 48 hours post-transfection and the luciferase activity was measured. Bar graphs represent two independent experiments, and show that miR-29 targets the ETO 3’UTR as indicated by a significant decrease in luciferase activity ( p > 0.01) C. SKNO-1 cells expressing the indicated miRs were harvested for western blot analysis 5 days post infection. Protein levels for ETO were determined by antibodies from two independent sources (see Materials and Methods for details). Levels of CDK6, a known target of miR-29a, were also examined. Tubulin was used as a loading control. Western blot represents results from two biological replicates.

Article Snippet: Following antibodies were used in this study: RUNX1 (Cat. No. 4334, Cell Signaling Technology, Inc., Danvers, MA), ETO (Cat. No. 4498S, Cell Signaling Technology, Inc.), RUNX1T1/ETO (Cat. No. ab124269; Abcam, Cambridge, MA), CDK6 Clone DCS83 (Cat. No. 3136S; Cell Signaling Technology, Inc.), Tubulin Clone DM1A (Cat. No. T9026; Sigma-Aldrich, St. Louis, MO), and normal (Cat. No. sc-2027 for anti-rabbit; Cat. No. sc-2025 for anti-mouse) and HRP-conjugated IgG (Cat. No. sc-2004 for anti-rabbit; Cat. No. sc-2005 for anti-mouse) antibodies (Santa Cruz Biotechnology, Dallas, TX).

Techniques: Sequencing, Transfection, Luciferase, Construct, Activity Assay, Expressing, Western Blot, Infection, Control

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: RUNX1T1-coding sequence (RUNX1T1-CDS) primers were designed for amplifying the open reading frame (ORF) of RUNX1T1 ( a ) and the sketch of RUNX1T amplification product from Hu lamb ( b ). The four lanes in the red bracket represented RUNX1T1 amplification product using subcutaneous fat cDNA ( a ). The dashed line represents the 5′ untranslated region of RUNX1T1 ( b ) .

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Sequencing, Amplification

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: The cDNA fragment and predicted protein sequences of ovine RUNX1T1. The start and stop codons are framed in red. The protein sequence was predicted by the BLAST tool of DNAMAN (version 6.0, LynnonBiosoft, San Ramon, CA, USA) and is presented under the coding sequence. All sequences were aligned by the DNAMAN (version 6.0, LynnonBiosoft) software. * means the translation is terminated.

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Sequencing, Software

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: Comparison of the bovine, mouse, and human RUNX1T1 amino acid sequences. The RUNX1T1 amino acid sequence (Accession No.: MH063277) was aligned to those of bovine (Accession No.: NP_001092855), murine (Accession No.: EDL05616), and human (Accession No.: NP_001185608.1) by DNAMAN. The same amino acid residues in four, three and two species are highlighted in red, green, yellow respectively, while the amino acid residues present in only one species are highlighted in white. The apostrophes indicates that these amino acid sequences are absent.

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Comparison, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: Expression pattern of RUNX1T1 isoforms in different tissues from 6-month-old Hu sheep. ( a ) Primer I was designed for identifying RUNX1T1 isoforms, primer II was used for qPCR of RUNX1T1-L mRNA. The red line represents the region which is present in the long isoform and absent in the short isoform. Depending on isoforms, bands of either 456 bp or 211 bp can be detected ( b ). Real-time PCR of mRNA for RUNX1T1-L (178 bp; ( c )) and FTO ( f ) in different tissues. Expression of the RUNX1T1-S (211 bp) in different tissues were determined by semi-qRT-PCR ( d , e ). Expression of gene was normalized to that of glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ), and relative to the expression in brain. Quantitative data are represented as the mean ± SEM ( n = 5).

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: Expression patterns of RUNX1T1 isoforms in the subcutaneous fat. The temporal expression of RUNX1T1 isoforms ( a – c ) and FTO mRNA ( d ) and RUNX1T1 protein ( e ) in the subcutaneous fat of Hu sheep at different ages were measured using Real-time PCR and Western blot (5 d, 3 M, 6 M and 24 M). d means day, M means month. Semi-quantitative analysis of the relative RUNX1T1 protein expression levels in the subcutaneous fat at different developmental stages was undertaken ( f ). Real-time PCR was performed on different developmental stages of subcutaneous fat to determine the expression level of marker genes ( g ), and immunofluorescence localization of RUNX1T1 in the subcutaneous fat of 24-month-old Hu sheep ( h – j ). Blue color indicates 4′,6-diamidino-2-phenylindole (DAPI) staining of the nuclei ( h ). Red color indicates the expression of RUNX1T1 ( i ). Pictures in ( g ) and ( h ) were merged ( j ). Scale bars = 100 µm. One-way ANOVA, and bars with different letters are significantly different ( p < 0.05). Quantitative data are shown as mean ± SEM ( n = 5).

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Marker, Immunofluorescence, Staining

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: Expression patterns of RUNX1T1 isoforms during ovine preadipocyte differentiation. The temporal expression pattern of RUNX1T1 isoforms and FTO during ovine preadipocyte differentiation ( a , b ); image of newly isolated and adipogenic differentiated ovine preadipocytes ( c ). Scale bars = 100 µm. One-way ANOVA followed by comparison of all time points to day 0 with Tuckey post hoc analysis, * p < 0.05. Quantitative data are represented as the mean ± SEM ( n = 4).

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Expressing, Isolation, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Characterization of RUNX1T1 , an Adipogenesis Regulator in Ovine Preadipocyte Differentiation

doi: 10.3390/ijms19051300

Figure Lengend Snippet: Adipogenic differentiation of ovine preadipocytes with RUNX1T1-L knockdown. siRUNX1T1-L and siNC were introduced into ovine preadipocytes when the density reached about 70%, and 48 h later, cells were subject to adipogenic differentiation ( a ). The expression of RUNX1T1-L was determined at 48 h post-transfection by Real-time PCR. Moreover, the expression of RUNX1T1-S was analyzed by semi-qRT-PCR ( b ). Ovine preadpocytes were transfected with siRUNX1T1-L or siNC and differentiated into mature adipocytes ( c ). Scale bars = 100 µm. After 8 days of differentiation, intracellular lipid droplets were stained using Oil Red-O. Lipid content was indirectly determined by measuring the optical density (OD) value at 510 nm on a spectrophotometer ( d ). Real-time PCR was performed on day 6 of adipogenic differentiation to determine the mRNA level of maker genes ( e ). Independent Students’ t -test. * p < 0.05 against siNC. Quantitative data are represented as the mean ± SEM ( n = 4).

Article Snippet: The frozen sections were fixed with 4% ( v / v ) paraformaldehyde for 1 h at 4 °C, washed three time with PBS, and then permeabilized with 0.2% ( v / v ) Triton X-100/PBS for 15 min. After treating with 3% ( w / v ) bovine serum albumin (BSA)/PBS for 30 min at room temperature, frozen sections were incubated overnight at 4 °C with primary rabbit anti-RUNX1T1 antibody (Proteintech) diluted 1:100 in 1% ( w / v ) BSA/PBS.

Techniques: Knockdown, Expressing, Transfection, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Staining, Spectrophotometry

Journal: Nature Communications

Article Title: CBFA2T3-GLIS2 mediates transcriptional regulation of developmental pathways through a gene regulatory network

doi: 10.1038/s41467-024-53158-9

Figure Lengend Snippet: A Co-immunoprecipitations using fusion-positive leukemic megakaryoblasts confirm the association of ETO, CtBP1, and p300 with the fusion. All immunoprecipitations were repeated twice, representative blots are shown. Source data are provided as a Source Data file. B CUT&RUN-seq has performed in fusion-positive megakaryoblasts from two secondary transplants and fusion negative megakaryoblasts cultured in vitro for ETO, CtBP1, and p300. Distribution of CUT&RUN-seq peak locations for ETO, CtBP1, and p300. CBFA2T3-GLIS2 is included as a comparison. TSS transcription start site, TTS transcription termination site, UTR untranslated region. Source data are provided as a Source Data file. C Summary of ETO, CtBP1, and p300 bound genes retained, gained, and lost in fusion-positive megakaryoblasts compared to fusion-negative megakaryoblasts. Source data are provided as a Source Data file. D Heatmaps of enrichment for CBFA2T3-GLIS2 (CG-TY), H3K27ac, ETO, CtBP1, and p300 at genes bound by the fusion. E Venn diagram showing the overlap of genes bound at the promoters of ETO, CtBP1, and p300 exclusively in fusion-positive megakaryoblasts and their overlap with fusion-bound genes (Supplementary Data ).

Article Snippet: Cell-bound beads were collected and re-suspended in Antibody Buffer and 0.5 ug of one of the following antibodies: TY1 (GenScript A01004-40), ETO (Bethyl A303-509A), CtBP1 (ThermoFisher 10972-1-AP), p300 (ThermoFisher PA1848), H3K27ac (Active Motif 39034), and rabbit IgG (Epicypher 13-0042).

Techniques: Cell Culture, In Vitro, Comparison

Journal: Nature Communications

Article Title: CBFA2T3-GLIS2 mediates transcriptional regulation of developmental pathways through a gene regulatory network

doi: 10.1038/s41467-024-53158-9

Figure Lengend Snippet: A–E Co-immunoprecipitation using 293T cells transfected with empty vector (MIG), wild-type fusion, or a fusion containing one or more of the mutations outlined in Fig. . All immunoprecipitations were repeated twice, representative blots are shown. Source data for all blots are provided as a Source Data file. A Immunoprecipitation of ETO followed by staining with the TY-1 tag to detect CBFA2T3-GLIS2 and the reciprocal immunoprecipitation of CBFA2T3-GLIS2 via the TY-1 tag followed by staining for ETO is shown. B TY-1-tagged wild-type CBFA2T3-GLIS2 and one of three FLAG-tagged mutant constructs were co-transfected into 293T cells to evaluate the ability of the mutant constructs to dimerize with the wild-type fusion. Immunoprecipitation by TY-1 followed by staining for FLAG and the reciprocal immunoprecipitation of FLAG followed by staining for TY-1 is shown. C 293T cells were co-transfected with two mutant constructs, one FLAG-tagged and one TY-1-tagged, to verify the results shown in ( B ). Immunoprecipitation of TY-1-tagged NHR2 deletion mutant followed by staining for FLAG again revealed a reduction of dimerization. D TY-1-tagged wild-type and mutant fusion constructs were transfected into 293T cells. Cells were immunoprecipitated for CtBP1 followed by staining for the fusion via TY-1 and the reciprocal immunoprecipitation of the fusion followed by staining for CtBP1. E TY-1-tagged wild-type and mutant fusion constructs were transfected into 293T cells. Cells were immunoprecipitated for p300, followed by staining for the fusion via TY-1, and the reciprocal immunoprecipitation of the fusion followed by staining for p300. F , G Transplantation of immunodeficient NSG-SGM3 mice with primary megakaryoblasts transduced with the mutant fusion constructs ( N = 5 per mutant construct, N = 9 wild type in panel F and 12 wild type in panel G ). p < 0.0001 for NHR2 deletion and NHR1-2 (DL380,487AS) mutant constructs compared to wild type. p -values determined by log-rank test. Source data are provided as a Source Data file.

Article Snippet: Cell-bound beads were collected and re-suspended in Antibody Buffer and 0.5 ug of one of the following antibodies: TY1 (GenScript A01004-40), ETO (Bethyl A303-509A), CtBP1 (ThermoFisher 10972-1-AP), p300 (ThermoFisher PA1848), H3K27ac (Active Motif 39034), and rabbit IgG (Epicypher 13-0042).

Techniques: Immunoprecipitation, Transfection, Plasmid Preparation, Staining, Mutagenesis, Construct, Transplantation Assay, Transduction

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: Genetic alterations in two c‐SCLC cases

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Amplification

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: RUNX1T1 expression is upregulated in the SCLC component of c‐SCLC tumors. (a) Examples of RNAscope in situ hybridization results of cores from a c‐SCLC tumor: two from the SCLC component (upper panel) and two from the NSCLC component (lower panel). Scale bar = 50 µm. RUNX1T1 mRNA signal is detected as red dots and MYC by the green dots. (b) Quantitation of the RUNX1T1 RNAscope signal of NSCLC and SCLC components (3 images/core for 2 cores of each histology) using CellProfiler. * P = 0.0292 from unpaired t‐test. Error bars represent mean ± SD. (c) INSM1 IHC stain of representative NSCLC and SCLC cores from c‐SCLC patient. Scale bar = 50 µm.

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Expressing, RNAscope, In Situ Hybridization, Quantitation Assay, Staining

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: RUNX1T1 is highly expressed in SCLC. (A) RUNX1T1 mRNA levels in cancer cell lines based on Affymetrix data from the CCLE. SCLC is highlighted with a red arrow and NSCLC with a blue arrow. (B) Western blot of RUNX1T1 expression in eight SCLC cell lines (DMS79, H2171, H1694, SHP77, H82, H446, H69, and SW1271) and four NSCLC cell lines (A549, H1299, H1650, and PC9); beta‐actin was used as the loading control. (C) RUNX1T1 mRNA levels in tumor samples detected by RNA‐Seq comparing SCLC with other cancers (BRCA: breast cancer, CR: colorectal cancer, GBM: glioblastoma, LUAD: lung adenoma NSCLC, LUSC: lung squamous NSCLC, PRAD: prostate adenoma cancer, SKCM: skin melanoma cancer). (D) RUNX1T1 in situ hybridization examples using RNAscope on clinical tumor samples of ‘pure’ SCLC, lung adenocarcinoma and lung squamous cell carcinoma. Red dots identify RUNX1T1 mRNA signal, green dots MYC mRNA signal. Scale bar = 50 µm.

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Western Blot, Expressing, Control, RNA Sequencing, In Situ Hybridization, RNAscope

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: Overexpression and knockout of RUNX1T1 lead to significant changes in hallmark pathways. (A) RUNX1T1 RT‐qPCR validating overexpression efficiency in cell lines ( n = 3 replicates of individual experiment/cell, error bars represent mean ± SEM). The house keep gene beta‐actin was used for normalization. No signal was detected in parental A549 and H1650 cells. (B) Western blot results validating RUNX1T1 overexpression (OE) and knockout (KO) in various cell lines. (C) Significantly changed hallmark pathways identified by GSEA after RUNX1T1 overexpression in NSCLC cell lines (H1299 and H1650) or RUNX1T1 knockout in SCLC cell lines (H82 and H2171). Blue and red bars show the overlapped pathways inversely affected by overexpression and knockout, respectively. NES: normalized enrichment score. (D) GSEA plots showing ‘E2F TARGETS’ genes depleted by RUNX1T1 overexpression in NSCLC (H1650 and H1299) and enriched by RUNX1T1 knockout in SCLC (H82 and H2171).

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Over Expression, Knock-Out, Quantitative RT-PCR, Western Blot

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: RUNX1T1 overexpression increases E2F activity and decreases CDKN1A (p21) expression. (A) E2F luciferase reporter assays in RUNX1T1 overexpressing cell lines A549, H1299, H841, and SW1271 ( n = 4 replicates of individual experiment/cell, 2 experiments/cell). Error bars represent mean ± SD. Significance by unpaired multiple t‐test. (b) E2F luciferase activity in H841 and SW1271 cells with stable knockdown of RB1 by shRNA ( n = 4 replicates of individual experiment/cell). Error bars represent mean ± SD. Significance by unpaired t‐test (c) CDKN1A (p21) western blot and RT‐qPCR ( n = 3 replicates of individual experiment/cell, error bars represent mean ± SEM) in RUNX1T1 overexpressing (OE) cells compared with the corresponding control. Beta‐actin was used as the loading control in western blotting and for normalization in qPCR. Significance by unpaired t ‐test.

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Over Expression, Activity Assay, Expressing, Luciferase, Knockdown, shRNA, Western Blot, Quantitative RT-PCR, Control

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: RUNX1T1 overexpression decreases histone acetylation at the CDKN1A (p21) promoter. (A) UCSC browser tracks showing CDKN1A gene annotation and precise transcription start site (TSS), as shown in GRO‐cap tracks of two different cell lines. DNaseI hyposensitivity and transcription factor ChIP‐seq tracks from ENCODE indicate tight regulation of the p21 gene. Enrichment of H3K27 ac (ENCODE ChIP‐seq data) and location of primer sets used in ChIP‐qPCR analysis are indicated. (B) RUNX1T1 ChIP‐qPCR results in overexpressing (OE) vs control H1299 and SW1271 cells targeting seven CDKN1A (p21) promoter and distal regions ( n = 3 replicates of biological duplicates/cell). Error bars represent mean ± SD (C) ChIP‐qPCR of pan‐histone 3 acetylation in OE vs control H1299 and SW1271 cells targeting the same seven CDKN1A (p21) promoter and distal regions ( n = 3 replicates of biological duplicates/cell). Error bars represent mean ± SD. (D) Western blot of CDKN1A (p21) in H1299 and SW1271 under Trichostatin A (TSA) treatment at 400nM and 2µM for six hours. Beta‐actin was used as the loading control. UT: untreated. DMSO: DMSO vehicle control.

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Over Expression, ChIP-sequencing, ChIP-qPCR, Control, Western Blot

Journal: Molecular Oncology

Article Title: Identification of RUNX1T1 as a potential epigenetic modifier in small‐cell lung cancer

doi: 10.1002/1878-0261.12829

Figure Lengend Snippet: RUNX1‐RUNX1T1 (AML/ETO) knockdown increases global H3K9 acetylation. Heatmap (A) and metagene plot (B) showing the enrichment of H3K9ac near the TSS of 21,364 curated genes in Kasumi‐1 cells (H3K9ac ChIP‐seq dataset SRR203377 and SRR203378). (C) H3K9ac level at CDKN1A promoter proximal region is shown using IGV (integrated genome viewer).

Article Snippet: Primary antibodies used were RUNX1T1 (Novus Biologicals, Centennial, CO, USA, #NBP2‐55747), p21 Waf1/Cip1 (Cell Signaling Technology, Danvers, MA, USA, #2947), and beta‐actin (Sigma, St. Louis, MO, USA, #A‐5441).

Techniques: Knockdown, ChIP-sequencing

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: C/EBPβ regulates homeostatic and oncogenic gastric cell proliferation

doi: 10.1007/s00109-016-1447-7

Figure Lengend Snippet: Genes from the C/EBPβ-clustered intestinal-type gastric cancer genes, showing their regulation in both intestinal-type tumors and C/EBPβ KO stomachs. Down-regulated genes in intestinal-type gastric cancer are up-regulated in the C/EBPβ KO stomach

Article Snippet: RUNX1t1 expression plasmid (pCMV-3xFlag-ETO) was obtained from ADDGENE® (ref: #12507).

Techniques:

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: C/EBPβ regulates homeostatic and oncogenic gastric cell proliferation

doi: 10.1007/s00109-016-1447-7

Figure Lengend Snippet: a Cross-species comparison of gene expression. Two-way hierarchical clustering was performed on the human gastric cancer samples using a strongly regulated gene cluster (shown in Supplementary Fig. ) from microarray-derived genes that differed between murine C/EBPβ KO and WT stomach ( p ≤ 0.01, FC ≥ 1.5). Depicted are the resultant gene and sample dendrograms and the corresponding expression intensity heatmap. The black box indicates a tumor cluster in which most of the genes show down-regulation ( bluish spots ). This tumor group consisted of 16 of the original 59 (≈27 %) samples and contained primarily cancers of the intestinal histological type. b Transfection of C/EBPβ isoforms LAP*, LAP, and LIP into gastric cell lines MKN28 and MKN45 repressed RUNX1t1 expression as measured by quantitative PCR. All bar graphs represent the result of at least three independent measurements; asterisk indicates p < 0.001

Article Snippet: RUNX1t1 expression plasmid (pCMV-3xFlag-ETO) was obtained from ADDGENE® (ref: #12507).

Techniques: Comparison, Gene Expression, Microarray, Derivative Assay, Expressing, Transfection, Real-time Polymerase Chain Reaction

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: C/EBPβ regulates homeostatic and oncogenic gastric cell proliferation

doi: 10.1007/s00109-016-1447-7

Figure Lengend Snippet: RUNX1t1 and gastric cancer. a RUNX1t1 expression was evaluated by immunohistochemistry in 64 human gastric cancer samples, and staining was classified by comparison to the expression in the normal mucosa ( left panel ). Thirty-eight percent of the cases showed reduced expression of nuclear RUNX1t1 (Tumor 1–3) in comparison to staining in the normal epithelium. b In 10 gastric tumors with reduced RUNX1t1, RNA levels were examined for C/EBPβ expression by qPCR. Only 3 out of 10 cases showed higher C/EBPβ expression as compared to WT. c The methylation status of the RUNX1t1 promoter was evaluated by methylation-specific PCR. Bisulfite treatment of tumor DNA converts unmethylated but not methylated cytosines to uracil, and subsequent methylation-specific PCR detects either methylated ( M ) or unmethylated ( U ) DNA. Fifty percent of the analyzed human gastric cancer cases (rows a-b, columns 1–5) present RUNX1t1 promoter hypermethylation. An increase in the methylation status is considered when the PCR product with methylation-specific primers is more intense than the one produced by non-methylated specific primers. d Ectopic expression of RUNX1t1 in MKN28 and MKN45 gastric cancer cell lines reduces gastric cancer cell proliferation as measured by BrdU incorporation assay. S-phase percentages are indicated in the FACS plots

Article Snippet: RUNX1t1 expression plasmid (pCMV-3xFlag-ETO) was obtained from ADDGENE® (ref: #12507).

Techniques: Expressing, Immunohistochemistry, Staining, Comparison, Methylation, Produced, BrdU Incorporation Assay

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: C/EBPβ regulates homeostatic and oncogenic gastric cell proliferation

doi: 10.1007/s00109-016-1447-7

Figure Lengend Snippet: RUNX1t1 modulates C/EBPβ activity. a Immunostaining shows C/EBPβ and RUNX1t1 colocalization in the neck zone of the normal human gastric mucosa. b Flag-Immunoprecipitation after transfection of gastric cell lines with a flag-tagged RUNX1t1 pulls down C/EBPβ. Visible in the input Western blot is also that RUNX1t1 does not affect C/EBPβ expression. c EMSA using a radiolabeled C/EBPβ consensus probe shows that transfection of RUNX1t1 to gastric cancer cells reduces the binding of C/EBPβ to DNA in MKN28 and MKN45 cell lines. Arrow indicated the super-shift. Also visible in the control Western blots, RUNX1t1 has no effect on basal C/EBPβ expression. d A RUNX1t1-dependent increase in the expression of TFF1 was visible by real-time PCR in the MKN74 cell line. e Luciferase assay in MKN45 cells transfected with the TFF1-luciferase fused promoter shows that co-transfection with RUNX1t1 reverts the repressive potential of C/EBPβ on the TFF1 promoter

Article Snippet: RUNX1t1 expression plasmid (pCMV-3xFlag-ETO) was obtained from ADDGENE® (ref: #12507).

Techniques: Activity Assay, Immunostaining, Immunoprecipitation, Transfection, Western Blot, Expressing, Binding Assay, Control, Real-time Polymerase Chain Reaction, Luciferase, Cotransfection