Journal: Haematologica

Article Title: Accumulation of hypoxia-inducible factor-1 alpha protein and its role in the differentiation of myeloid leukemic cells induced by all-trans retinoic acid.

doi: 10.3324/haematol.13096

Figure Lengend Snippet: Figure 5. Specific suppression of Runx1, C/EBPα and PU.1 signifi- cantly prevents HIF-1α-triggered differentiation as well as its cooperation with ATRA. U937Tclone cells were stably transfected with a negative control (NC) or shRNA against Runx1 (shR-A2), C/EBPα (shR-C2), or PU.1 (shR-P3). The transfected cells were then incu- bated in the presence or absence of tetracycline and/or 10-8M of ATRA for 24 hours before blotting proteins as indi- cated (A) and for 3 days to detect CD11b+% cells (B).

Article Snippet: Western blot Cell lysates were fractionated on a 10% sodium dodecyl sulfate–polyacrylamide gel and immunoblots were performed with primary antibodies against HIF1α and HIF-1β (BD Transduction, San José, CA, USA), C/EBPα, Runx1 and PU.1 (Santa Cruz, CA, USA).

Techniques: Stable Transfection, Transfection, Negative Control, shRNA

Journal: Bone Research

Article Title: Runx1 protects against the pathological progression of osteoarthritis

doi: 10.1038/s41413-021-00173-x

Figure Lengend Snippet: The expression of target candidates in cartilage. a qPCR results showing changes in TAPT1 , RIC1 , and FGF20 in chondrocytes induced by siRunx1. b qPCR results showing changes in TAPT1 , RIC1 , and FGF20 in chondrocytes induced by Runx1 overexpression. c , d IHC staining showing AAV-Runx1 overexpression-induced changes in TAPT1 expression in articular cartilage at 12 weeks after ACLT surgery ( a ) and the growth plate at 24 weeks after ACLT surgery ( b ). e – f IHC staining showing AAV-Runx1 overexpression-induced changes in RIC1 expression in articular cartilage at 12 weeks after ACLT surgery ( a ) and the growth plate at 24 weeks after ACLT surgery ( b ). g – h IHC staining showing AAV-Runx1 overexpression-induced changes in FGF20 expression in articular cartilage at 12 weeks after ACLT surgery ( a ) and the growth plate at 24 weeks after ACLT surgery ( b ). i Optical density quantitative analysis showing changes in TAPT1 expression in articular cartilage and the growth plate. j Optical density quantitative analysis showing changes in RIC1 expression in articular cartilage and the growth plate. k Optical density quantitative analysis showing changes in FGF20 expression in articular cartilage and the growth plate. These results are based on at least three independent experiments ( n = 3). All significance data presented in a , b , i , j , and k are based on two-tailed Student’s t -tests

Article Snippet: We used siRunx1 (mouse, sc-37678, Santa Cruz Biotechnology, Delaware Avenue, CA) and siRNA-A Control (sc-37007) to knock down Runx1, and lentivirus transfection of Runx1 was performed to overexpress Runx1. qPCR was performed to examine changes in genes induced by Runx1 knockdown or overexpression.

Techniques: Expressing, Over Expression, Immunohistochemistry, Two Tailed Test

Journal: Journal of Virology

Article Title: RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression

doi: 10.1128/JVI.00943-20

Figure Lengend Snippet: Putative RUNX CBSs are more enriched and more closely localized to the TSSs of viral genes in HSV-1 and HSV-2 genomes than would be expected by chance. (A) Normalized RNA-Seq expression of Runx1, Runx3, and Ctcf mRNA in longitudinal muscle myenteric plexus (LMMP) and dorsal root ganglion (DRG) neurons of B6 mice from the data set previously published by Khoury-Hanold et al. (9). The data are means ± the standard errors (SE; n = 2). (B) Quantification of observed and expected putative RUNX consensus binding sites (CBS) in HSV-1 and HSV-2 genomes. The sequences utilized for CBS analysis are listed in Table 1. The expected numbers were calculated accounting for each genome’s GC content. (C) Average distance between TSS of viral genes and closest CBS from actual set of binding sites (blue line) and 1,000 simulated random distributions of CBSs (red curve).

Article Snippet: Human RUNX1 (SC123977), RUNX3 (SC302527), and CBFβ (SC110848) cDNA clones were purchased from OriGene. pIRES2-AcGFP1 plasmid (632435) was purchased from Clontech.

Techniques: RNA Sequencing, Expressing, Binding Assay

Journal: Journal of Virology

Article Title: RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression

doi: 10.1128/JVI.00943-20

Figure Lengend Snippet: Overexpression of RUNX1, but not RUNX3, represses HSV-1 viral gene expression. Validation of RUNX (A) mRNA and (B) protein in stable cell lines overexpressing empty vector (Emp), RUNX1 (R1), DNA-binding mutant D198G RUNX1 (R1D), or RUNX3 (R3). (C) After Emp, R1, and R3 cell lines were infected with HSV-1 at an MOI of 1 for 48 h, the mRNA expression of viral genes was measured by RT-qPCR and normalized to HPRT mRNA. (D) The same experiment as in panel C was repeated for Emp, R1, and R1D cell lines. The data are means ± the SE (n = 3). *, P < 0.05; ***, P < 0.01; ****, P < 0.001; *****, P < 0.0001 (as assessed by two-way ANOVA).

Article Snippet: Human RUNX1 (SC123977), RUNX3 (SC302527), and CBFβ (SC110848) cDNA clones were purchased from OriGene. pIRES2-AcGFP1 plasmid (632435) was purchased from Clontech.

Techniques: Over Expression, Gene Expression, Biomarker Discovery, Stable Transfection, Plasmid Preparation, Binding Assay, Mutagenesis, Infection, Expressing, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression

doi: 10.1128/JVI.00943-20

Figure Lengend Snippet: RUNX1 overexpression blunts HSV-1 viral gene transcription over time. After Emp, R1, and R3 cell lines were infected with HSV-1 at an MOI of 1, the mRNA expression of immediate early genes (A), early genes (B), and late genes (C) was measured by RT-qPCR and normalized to HPRT mRNA at 12, 24, 48, and 72 hpi. The data are means ± the SE (n = 3). ##, P < 0.1; *****, P < 0.0001 (as assessed by two-way ANOVA tests).

Article Snippet: Human RUNX1 (SC123977), RUNX3 (SC302527), and CBFβ (SC110848) cDNA clones were purchased from OriGene. pIRES2-AcGFP1 plasmid (632435) was purchased from Clontech.

Techniques: Over Expression, Infection, Expressing, Quantitative RT-PCR

Journal: Journal of Virology

Article Title: RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression

doi: 10.1128/JVI.00943-20

Figure Lengend Snippet: RUNX1 binds to putative binding sites upstream of HSV-1 viral genes. HEK293T cells transiently overexpressing empty vector (Emp), RUNX1 (R1), DNA-binding mutant D198G RUNX1 (R1D), or RUNX3 (R3) were infected with HSV-1 at an MOI of 10. After 5 h infection, nuclear DNA was immunoprecipitated with isotype IgG, anti-RUNX1, or anti-RUNX3 antibodies. After elution and purification of bound single-stranded DNA, RUNX enrichment at putative binding sites in the promoter regions of RS1 (A), UL8 (B), US6 (C), UL20 (D), and “RUNX desert region” (HSV Neg) (E) was assessed by ChIP-qPCR and normalized to input DNA. The data are means ± the SE (n = 3). *, P < 0.05; ***, P < 0.01; ****, P < 0.001 (as assessed by two-way ANOVA).

Article Snippet: Human RUNX1 (SC123977), RUNX3 (SC302527), and CBFβ (SC110848) cDNA clones were purchased from OriGene. pIRES2-AcGFP1 plasmid (632435) was purchased from Clontech.

Techniques: Binding Assay, Plasmid Preparation, Mutagenesis, Infection, Immunoprecipitation, Purification, ChIP-qPCR

Journal: Journal of Virology

Article Title: RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression

doi: 10.1128/JVI.00943-20

Figure Lengend Snippet: Overexpression of RUNX1, but not RUNX3, decreases HSV-1 infection in vitro. HEK293T cells overexpressing empty vector, RUNX1, and RUNX3 were infected with HSV-1 at an MOI of 1. (A) Cells were stained with anti-HSV-1 polyclonal antibody, and fluorescence was measured by flow cytometry at 12, 24, 48, and 72 hpi. Histograms are shown normalized to mode. Mock-infected cells are displayed in gray. Dotted line indicates cutoff for HSV-1+ cells. (B) Quantification of HSV-1 MFI at each time point. (C) Live proportion of singlets as measured by live-dead staining at each time point. (D and E) The same experiment as in panels A to C was performed with stable cell lines overexpressing R1D. (D) HSV-1 MFI was measured by flow cytometry at 48 hpi. (E) Viral titers of supernatant 48 hpi were measured by plaque assay. The data are means ± the SE (n = 3). *, P < 0.05; ***, P < 0.01; ****, P < 0.001; *****, P < 0.0001 (as assessed by two-way ANOVA and one-way ANOVA).

Article Snippet: Human RUNX1 (SC123977), RUNX3 (SC302527), and CBFβ (SC110848) cDNA clones were purchased from OriGene. pIRES2-AcGFP1 plasmid (632435) was purchased from Clontech.

Techniques: Over Expression, Infection, In Vitro, Plasmid Preparation, Staining, Fluorescence, Flow Cytometry, Stable Transfection, Plaque Assay

Journal: Journal of Virology

Article Title: RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression

doi: 10.1128/JVI.00943-20

Figure Lengend Snippet: RUNX1 knockdown in IMR-32 induces HSV-1 viral gene expression and promotes HSV-1 infection. After 24 h transfection with negative-control siRNA (siNC) or siRNA specific to RUNX1 (siRUNX1), IMR-32 neuroblastoma cells were infected with HSV-1 at an MOI of 1 for 48 h. (A) The mRNA expression of viral genes was measured by RT-qPCR and normalized to HPRT. (B and C) Validation of RUNX1 knockdown at (B) protein and (C) mRNA levels 24 h after transfection with siRNA. (D to E) Quantification of HSV MFI (D) and uninfected proportion of singlets (E). (F) Viral titers of HSV-1 present in supernatant 48 hpi was measured by plaque assay. The data are means ± the SE (n = 3). *, P < 0.05; ***, P < 0.01 (as assessed by two-way ANOVA and Student t test).

Article Snippet: Human RUNX1 (SC123977), RUNX3 (SC302527), and CBFβ (SC110848) cDNA clones were purchased from OriGene. pIRES2-AcGFP1 plasmid (632435) was purchased from Clontech.

Techniques: Knockdown, Gene Expression, Infection, Transfection, Negative Control, Expressing, Quantitative RT-PCR, Biomarker Discovery, Plaque Assay

Journal: Journal of Virology

Article Title: RUNX Binding Sites Are Enriched in Herpesvirus Genomes, and RUNX1 Overexpression Leads to Herpes Simplex Virus 1 Suppression

doi: 10.1128/JVI.00943-20

Figure Lengend Snippet: Cloning and mutagenesis primers

Article Snippet: Human RUNX1 (SC123977), RUNX3 (SC302527), and CBFβ (SC110848) cDNA clones were purchased from OriGene. pIRES2-AcGFP1 plasmid (632435) was purchased from Clontech.

Techniques: Cloning, Mutagenesis