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: The Journal of biological chemistry

Article Title: Lactate promotes neuronal differentiation of SH-SY5Y cells by lactate-responsive gene sets through NDRG3-dependent and -independent manners.

doi: 10.1016/j.jbc.2023.104802

Figure Lengend Snippet: Figure 11. TEAD1 and ELF4 differently affect lactate-NDRG3-regulated neuronal maker gene expressions in SH-SY5Y cells. A–C, the protein expression levels of NF-H, RUNX1T1 and SYT4 in siNDRG3 (A), siTEAD1 (B), and siELF4 (C) or scrambled siRNA transfected SH-SY5Y cells (n = 4). Relative protein levels were normalized to the levels of RPL13A and α-actinin in the same samples and presented as fold change to control. All data are reported as means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 versus control. Statistical significance was determined by unpaired two-tailed t test. Since these protein expression levels were analyzed by the same lysate in Figure 8C, the blots of RPL13a, α-actinin, and NDRG3 were the same and used for the normalization. D, a schematic illustration of lactate-induced neuronal differentiation mechanism through NDRG3-dependent and -independent signaling axis.

Article Snippet: The membranes were blocked in 5% fat-free milk in TBST (20 mM Tris-HCl (pH 7.6), 0.15 M sodium chloride, and 0.1% Tween 20) for 1 h, washed three times with TBST, and incubated with primary antibodies that probe for NSE (9536, CST), ID2 (3431, CST), MAP2 (4542, CST), NF-H (ab4680, abcam), RPL13A (2765, CST), α-actinin (6487, CST), Cyclophilin B (43603, CST), β-actin (8457, CST), NDRG3 (ab133715, abcam), MCT1 (20139-1-AP, Proteintech), MCT2 (ab224627, abcam), MCT4 (ab74109, abcam), TEAD1 (12292, CST), ELF4 (sc-390689, Santacruz), EEF1A2 (GTX102326, GeneTex), HES7 (AP9712a, Abcepta), TLE2 (GTX106107, GeneTex), RUNX1T1 (15494-1-AP, Proteintech) and SYT4 (12642-1-AP, Proteintech) in TBST for overnight.

Techniques: Expressing, Transfection, Control, Two Tailed Test

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: 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