Journal: Cells

Article Title: Transcriptomic Analysis of Adult Mouse Cardiac Stromal Cells Using Single-Cell qRT-PCR

doi: 10.3390/cells15040384

Figure Lengend Snippet: Single-cell analysis reveals enrichment of Tcf21 and low expression of Tbx5 in CTs compared to CDCs and CFs. ( A ) Expression of 43 genes was analysed at the single-cell level in the four cell populations shown: CTs ( n = 55), CDCs ( n = 41), and CFs ( n = 29) at passage 3, as well as HL-1 cardiomyocytes ( n = 13). Both CTs and CDCs represent 3 biological replicates. The heatmap illustrates expression as −∆CT values (blue indicates low or absent expression; red indicates high expression). Samples were ordered based on cell type, and the genes were grouped using a hierarchical clustering algorithm based on the underlying co-expression pattern. ( B ) Single-cell expression of the 43 genes tested, shown individually for the four cell populations, with the mean represented as a black diamond in each group.

Article Snippet: Tbx5 , Mm00803518_m1 , Ddr2 , Mm00445615_m1.

Techniques: Single-cell Analysis, Expressing, Single Cell

Journal: Cells

Article Title: Transcriptomic Analysis of Adult Mouse Cardiac Stromal Cells Using Single-Cell qRT-PCR

doi: 10.3390/cells15040384

Figure Lengend Snippet: PCA of the three cardiac stromal populations and HL-1 cardiomyocytes. ( A ) PCA of the four cell populations shown: CTs ( n = 55), CDCs ( n = 41), and CFs ( n = 29) at passage 3, as well as HL-1 cardiomyocytes ( n = 13). Both CTs and CDCs represent 3 biological replicates. Dim.1 separates the cardiac stromal populations, whereas Dim.2 shows a distinct separation between cardiac stromal and myocyte populations. Gene loadings contributing to each Dim suggest that a small subset of genes explains the cross-group variability captured by Dim.1 and Dim.2. Tcf21 is associated with CTs (emphasised by the blue circle), whereas Tbx5 is associated with CDCs and CFs (emphasised by the brown circle). The separation between cardiac stromal and myocyte populations is reflected by clustering of core cardiac and cardiomyocyte genes ( Nkx2-5 , Wif1 , Nppa , Myl2 , and Myh6 ), which are emphasised by the pink circle. ( B ) PCA of the three cardiac stromal populations alone: CTs ( n = 28), CDCs ( n = 41), and CFs ( n = 27). Both CTs and CDCs represent 3 biological replicates. Gene loadings contributing to each Dim show that Tcf21 is associated with CTs, whereas Tbx5 is associated with CDCs and CFs.

Article Snippet: Tbx5 , Mm00803518_m1 , Ddr2 , Mm00445615_m1.

Techniques:

Journal: Biochemical and biophysical research communications

Article Title: Noncanonical Notch signals have opposing roles during cardiac development.

doi: 10.1016/j.bbrc.2021.08.094

Figure Lengend Snippet: Fig. 1. Noncanonical Notch inhibits heart development in Mesp1þ CPCs. A. E8.5 embryos whole mount and transverse sectioning showing decreased heart size in NICDOE embryos, independent of RBP-j. Cardiac crescent (CC) develops normally, but looping heart (HT) with visible left ventricle (LV) and outflow tract (OT) are not present in mutant embryos (n ¼ 10). White scale bars represent 300 mm. B. Relative gene expression of FHF/SHF genes from embryos dissected at E8.0. FHF marker Tbx5 shows no significant difference and FHF marker Gata4 shows a slight upregulation in NICDOE embryos. SHF markers Six2 and Tbx1 both show significant downregulation in NICDOE embryos (n ¼ 9). C. Representative flow cytometric analysis plots quantifying GFPþ/RFP þ cells after DAPT treatment. Percentage of RFPþ (SHF) cells significantly decreases with DAPT treatment. D. DAPT treatment in CPCs during induction leads to decrease in Wnt activity.

Article Snippet: Immunostainings were completed using Nkx2.5 antibody (Santa Cruz: sc-8697), Tbx5 antibody (Atlas Antibodies: HPA008786), and DAPI (Life Technologies).

Techniques: Mutagenesis, Gene Expression, Marker, Activity Assay

Journal: Biochemical and biophysical research communications

Article Title: Noncanonical Notch signals have opposing roles during cardiac development.

doi: 10.1016/j.bbrc.2021.08.094

Figure Lengend Snippet: Fig. 2. Noncanonical Notch increases heart size in Nkx2.5 CPCs. A. NICDOE embryos show increased heart size independent of RBP-j. Of note, LV is enlarged in NICDOE embryos compared to control (n ¼ 8/11; n ¼ 6/9). White scale bars represent 300 mm. B. Tbx5 staining reveals an expansion of LV in transverse sections of NICDOE embryos. White scale bars represent 300 mm. C. Ink injection experiments show disrupted circulation in NICDOE embryos independent of RBP-j (n ¼ 10/12).

Article Snippet: Immunostainings were completed using Nkx2.5 antibody (Santa Cruz: sc-8697), Tbx5 antibody (Atlas Antibodies: HPA008786), and DAPI (Life Technologies).

Techniques: Control, Staining, Injection

Journal: Current Research in Biotechnology

Article Title: Generation of a transducible version of a bioactive recombinant human TBX5 transcription factor from E. coli

doi: 10.1016/j.crbiot.2022.01.004

Figure Lengend Snippet: Fig. 2. In silico expression analysis of the TBX5 gene before and after codon optimization. (A) The bar graph represents the percentage distribution of codons before (red) and after (green) codon optimization in computed codon quality groups with GRCAT. Codons with a value ≤30 (threshold indicated by a dotted line) are more likely to decrease the gene expression in E. coli. (B) Bar graph showing the relative adaptiveness value of each codon (first 50 codons only) before (top) and after (bottom) codon optimization with GCUAT. A relative adaptiveness value ≤30 for a particular codon (arrow) indicates that it is most likely to decrease the gene expression in E. coli.

Article Snippet: For identification of rhTBX5 fusion proteins, antibodies to histidine (1:5000, BB‐AB0010, BioBharati) and Tbx5 (1:2000, sc‐ 515536, Santa Cruz Biotechnology) were used.

Techniques: In Silico, Expressing, Gene Expression

Journal: Current Research in Biotechnology

Article Title: Generation of a transducible version of a bioactive recombinant human TBX5 transcription factor from E. coli

doi: 10.1016/j.crbiot.2022.01.004

Figure Lengend Snippet: Fig. 3. Schematic representation of the tagging pattern in the human TBX5 gene and verification of cloning. (A) Pictorial representation of TBX5 fusion gene constructs along with restriction sites. (B) Restriction digestion analysis of recombinant plasmids bearing TBX5 fusion gene inserts. His, polyhistidine tag (8 × ); TAT, transactivator of transcription; NLS, nuclear localization signal.

Article Snippet: For identification of rhTBX5 fusion proteins, antibodies to histidine (1:5000, BB‐AB0010, BioBharati) and Tbx5 (1:2000, sc‐ 515536, Santa Cruz Biotechnology) were used.

Techniques: Cloning, Construct, Recombinant

Journal: Current Research in Biotechnology

Article Title: Generation of a transducible version of a bioactive recombinant human TBX5 transcription factor from E. coli

doi: 10.1016/j.crbiot.2022.01.004

Figure Lengend Snippet: Fig. 4. Analysis of the solubility of rhTBX5 fusion proteins. The protein samples were resolved on 12% SDS-PAGE gels with normalized loading; either Coomassie staining or immunoblotting with anti-histidine was performed. (A) rhHTN-TBX5 (n = 7) and (B) rhTBX5-NTH (n = 5). M, protein marker (kDa); L, total cell lysate; P, insoluble pellet fraction; S, soluble supernatant fraction; Ab, antibody; * truncated fragments of rhTBX5 fusion protein.

Article Snippet: For identification of rhTBX5 fusion proteins, antibodies to histidine (1:5000, BB‐AB0010, BioBharati) and Tbx5 (1:2000, sc‐ 515536, Santa Cruz Biotechnology) were used.

Techniques: Solubility, SDS Page, Staining, Western Blot, Marker

Journal: PloS one

Article Title: Cardiac gene activation analysis in mammalian non-myoblasic cells by Nkx2-5, Tbx5, Gata4 and Myocd.

doi: 10.1371/journal.pone.0048028

Figure Lengend Snippet: Figure 1. Nkx2-5, Tbx5, Gata4 and Myocd lentiviral vectors transactivated cis-element reporters containing cognate sequences of Nkx2-5, Tbx5, Gata4, and Srf binding sites. CV-1 cells were cotransfected with Tet-transactivator vector, corresponding luciferase reporters, and one or more of Nkx2-5, Tbx5, Gata4, Myocd, or LacZ tetracycline-dependent lentiviral vectors. Dual-luciferase assays were performed 48 hours after doxycycline induction. (A) TRE-Nkx2-5-IPR transactivated NKE cis-element reporter. (B) TRE-Tbx5-INR transactivated TBE cis-element reporter. (C) TRE- Gata4-IPG transactivated GATA cis-element reporter. (D) TRE-Myocd-ING transactivated SRE cis-element reporter in the presence of Srf. Each experiment was performed in quadruplicate. Blank, without the addition of tetracycline-dependent lentiviral vector; TRE-IPR, TRE-INR, TRE-IPG, and TRE-ING are empty vector controls for Nkx2-5, Tbx5, Gata4, and Myocd lentiviruses. TRE-LacZ-IPR, TRE-LacZ-INR, TRE-LacZ-IPG, and TRE-LacZ-ING are LacZ vector controls for Nkx2-5, Tbx5, Gata4, and Myocd lentiviruses. TRE-N-IPR, TRE-T-INR, TRE-G-IPG, and TRE-M-ING are Nkx2-5, Tbx5, Gata4, and Myocd lentiviruses. *P,0.01 vs LacZ groups. doi:10.1371/journal.pone.0048028.g001

Article Snippet: Briefly, complementary deoxyribonucleic acids (cDNAs) of human Nkx2-5 (OriGene) and peroxisome Discosoma sp. red fluorescent protein variant MST (DsRed-MST), human Tbx5 (OriGene) and nuclear DsRed-MST, human Gata4 (OriGene) and peroxisome enhanced green fluorescent protein (EGFP), and human Myocd (accession number AY764180, provided by Dr. Antoine A.F. de Vries) [22] and nuclear EGFP were linked together with an encephalomyocarditis virus-derived IRES sequence, respectively.

Techniques: Binding Assay, Plasmid Preparation, Luciferase

Journal: PloS one

Article Title: Cardiac gene activation analysis in mammalian non-myoblasic cells by Nkx2-5, Tbx5, Gata4 and Myocd.

doi: 10.1371/journal.pone.0048028

Figure Lengend Snippet: Figure 2. Tbx5+Myocd and Tbx5+Gata4+Myocd combinations induced Myh6 and Tnnt2 cardiac marker protein expression in 10T1/ 2 non-myoblastic cells. 10T1/2 fibroblasts were infected with Nkx2-5, Tbx5, Gata4, Myocd lentivirus alone, or different combinations. LacZ lentivirus was used as control. Ten days after doxycycline induction, Myh6 and Tnnt2 protein expression was examined by incubating the cells with Myh6 or Tnnt2 primary antibody and was visualized by Alexa Fluor 350-conjugated secondary antibody. Only T+M and T+G+M combinations induced de novo Myh6 and Tnnt2 protein expression. Myh6+ and Tnnt2+ cell induction rates in designated cell populations were summarized in Table 1. Insets showed nascent fibril-like structures. Scale bar = 20 mm. N, Nkx2-5; T, Tbx5; G, Gata4; M, Myocd. doi:10.1371/journal.pone.0048028.g002

Article Snippet: Briefly, complementary deoxyribonucleic acids (cDNAs) of human Nkx2-5 (OriGene) and peroxisome Discosoma sp. red fluorescent protein variant MST (DsRed-MST), human Tbx5 (OriGene) and nuclear DsRed-MST, human Gata4 (OriGene) and peroxisome enhanced green fluorescent protein (EGFP), and human Myocd (accession number AY764180, provided by Dr. Antoine A.F. de Vries) [22] and nuclear EGFP were linked together with an encephalomyocarditis virus-derived IRES sequence, respectively.

Techniques: Marker, Expressing, Infection, Control

Journal: PloS one

Article Title: Cardiac gene activation analysis in mammalian non-myoblasic cells by Nkx2-5, Tbx5, Gata4 and Myocd.

doi: 10.1371/journal.pone.0048028

Figure Lengend Snippet: Figure 3. Tbx5+Gata4+Myocd was the most effective activator of cardiac genes. 10T1/2 fibroblasts were infected with Tbx5, Gata4, Myocd lentivirus alone, or different combinations. LacZ lentivirus was used as control. Genetically unbiased genome-wide expression profiles were obtained 14 days after doxycycline induction. (A) Global gene expression. (B) Cardiac structural gene enrichment. (C) Smooth muscle gene enrichment. (D) Skeletal muscle gene enrichment. (E) Cardiac-related transcription factor enrichment. Signal values were log2 transformed. Signal values below 0 were set to 1. Each group included biological triplicates. T, Tbx5; G, Gata4; M, Myocd; TG, T+G; GM, G+M; TM, T+M; TGM, T+G+M. doi:10.1371/journal.pone.0048028.g003

Article Snippet: Briefly, complementary deoxyribonucleic acids (cDNAs) of human Nkx2-5 (OriGene) and peroxisome Discosoma sp. red fluorescent protein variant MST (DsRed-MST), human Tbx5 (OriGene) and nuclear DsRed-MST, human Gata4 (OriGene) and peroxisome enhanced green fluorescent protein (EGFP), and human Myocd (accession number AY764180, provided by Dr. Antoine A.F. de Vries) [22] and nuclear EGFP were linked together with an encephalomyocarditis virus-derived IRES sequence, respectively.

Techniques: Infection, Control, Genome Wide, Expressing, Gene Expression, Transformation Assay

Journal: Frontiers in physiology

Article Title: PLXND1-mediated calcium dyshomeostasis impairs endocardial endothelial autophagy in atrial fibrillation.

doi: 10.3389/fphys.2022.960480

Figure Lengend Snippet: FIGURE 1 Histological and cellular changes in the hearts of atrial fibrillation mouse models. (A), Experimental schema of AF mice model. Male Tbx5+/−mice (Tbx5fl/+; CMV-Cre) and CMV-Cre mice were used as SR and AF mice respectively. (B) ECG tracing of SR mice (upper) and AF mice (lower); rapid and irregular atrial waves are indicated by red arrows. (C) Histological images of hearts from SR and AF mice obtained by Masson’s trichrome staining. Scale bar: 100 μm (right panel). (D) The degree of fibrosis was evaluated and calculated by measuring the blue regions (collagen) relative to the total tissue area in sections, as shown in (C). (E) Representative images stained with WGA (red) to delineate the sarcolemma, and DAPI (blue). Scale bar, 20 μm. (F) Quantification of the CSAs of atrial cardiomyocytes. (G–I) Statistics of plasma concentrations of BNP, TnI, and CK-MB in mice in two groups. AF, atrial fibrillation; SR, sinus rhythm; ECG, electrocardiogram; WGA, wheat germ agglutinin; CSA, cross-sectional area; BNP, brain natriuretic peptide; TnI, troponin I; CK-MB, creatine kinase MB.

Article Snippet: Tbx5 fl/fl mice were mated with CMVCre transgenic mice (Cyagen Biosciences) to constitutively delete the sequences between loxP sites (KO region: ~3,240 bp).

Techniques: Staining, Clinical Proteomics

Journal: PLoS ONE

Article Title: Induced cardiomyocyte maturation: Cardiac transcription factors are necessary but not sufficient

doi: 10.1371/journal.pone.0223842

Figure Lengend Snippet: Neonatal cardiac fibroblasts were transfected with negmiR (control) or miR combo and siRNAs that target Gata4, Tbx5, Mef2C, and Hand2 (non-targeting siRNA used as a control). (A) Expression of cardiac commitment genes was assessed four days after transfection by qPCR. N = 5. *Comparisons between negmiR and miR combo in the control siRNA and GMTH siRNA groups: *** P<0.0001, **P<0.001, *P<0.05. †Comparisons between GMTH siRNA groups and negmiR + control siRNA group: †††P<0.001, ††P<0.01, †P<0.05. (B) Expression of cardiomyocyte mature marker genes was assessed fourteen days after transfection by qPCR. N = 3–5. *Comparisons between negmiR and miR combo in the control siRNA and GMTH siRNA groups: **P<0.001, *P<0.05, ns not significant.

Article Snippet: Gata4 (MR227022), Mef2C (MR207045) and Tbx5 (MR227369) expression plasmids were purchased from Origene, MD.

Techniques: Transfection, Control, Expressing, Marker

Journal: PLoS ONE

Article Title: Induced cardiomyocyte maturation: Cardiac transcription factors are necessary but not sufficient

doi: 10.1371/journal.pone.0223842

Figure Lengend Snippet: (A) MEF (N = 5) and neonatal cardiac fibroblasts (N = 4) were transfected with dCas9-VPR and validated gRNAs for Ascl1. Expression was assessed three days after transfection by qPCR and shown as a fold change compared to a control plasmid. (B) MEF cells (N = 5) were transfected with dCas9-VPR and 4 gRNAs for Gata4 and 5 gRNAs for Mef2C and Tbx5. After 3 days, commitment marker expression was determined by qPCR and shown as a fold change compared to the mock transfection. (C) Top panels: MEF cells (N = 2) were transfected with dCas9-VPR and various combinations of the original pool of gRNAs. After 3 days, commitment marker expression was determined by qPCR and shown as a fold change compared to the mock transfection. The arrows indicate the active gRNAs in each pool; i.e. gRNAs which when removed from the pool of gRNAs, prevent dCas9-VPR from inducing gene expression. In each pool, two active gRNAs were identified and these two gRNAs were used for subsequent experiments. Bottom panels: MEF cells (N = 3) were transfected with the top two guide-RNAs for Gata4, Mef2C and Tbx5. After 3 days, commitment marker expression was determined by qPCR and shown as a fold change compared to the mock transfection.

Article Snippet: Gata4 (MR227022), Mef2C (MR207045) and Tbx5 (MR227369) expression plasmids were purchased from Origene, MD.

Techniques: Transfection, Expressing, Control, Plasmid Preparation, Marker, Gene Expression

Journal: PLoS ONE

Article Title: Induced cardiomyocyte maturation: Cardiac transcription factors are necessary but not sufficient

doi: 10.1371/journal.pone.0223842

Figure Lengend Snippet: Neonatal cardiac fibroblasts were transfected with a combination of Gata4 RNA, Tbx5 RNA and Mef2C RNA (ratio empirically determined for maximal expression of maturation markers). (A) Expression of cardiac commitment genes was assessed four days after transfection by qPCR. N = 5. (B) Expression of cardiomyocyte mature marker genes was assessed fourteen days after transfection by qPCR. N = 5. *Comparisons were made between GMT RNA: **P<0.001, *P<0.05, ns not significant.

Article Snippet: Gata4 (MR227022), Mef2C (MR207045) and Tbx5 (MR227369) expression plasmids were purchased from Origene, MD.

Techniques: Transfection, Expressing, Marker