Journal: Stem Cell Reports

Article Title: SHOX2 Overexpression Favors Differentiation of Embryonic Stem Cells into Cardiac Pacemaker Cells, Improving Biological Pacing Ability

doi: 10.1016/j.stemcr.2014.11.004

Figure Lengend Snippet: Primer Sets for Quantitative PCR

Article Snippet: Mouse Tbx18 , Mm00470177_m1 , NM_023814.4 , chromosome 9: 87702800–87731260 , 6–7 , 64.

Techniques: Sequencing, Amplification

Journal: Stem cells and development

Article Title: Efficient Differentiation of TBX18 + /WT1 + Epicardial-Like Cells from Human Pluripotent Stem Cells Using Small Molecular Compounds.

doi: 10.1089/scd.2016.0208

Figure Lengend Snippet: FIG. 2. WNT and RA act synergistically to specify TBX18+/WT1+ cell fate. (A) qRT-PCR analysis of TBX18 and WT1 expression in D14 cultures following treatment with 5 mM CHIR and the indicated concentrations of RA. BMS493, 5 mM. Gene expression was normalized to TBP. Error bars represent SEM; n = 3; *p < 0.05, **p < 0.01 versus CHIR (5 mM)-treated cultures, analyzed by the Student’s t-test. (B) Flow cytometry analysis of the proportion of WT1+ cells in D14 cultures. Error bars represent SEM; n = 3. (C) High-content imaging assays of the proportion of TBX18+/WT1+ cells in D14 cultures treated with CHIR, RA/CHIR, or BMS493/CHIR. Error bars represent SEM; n = 3; **p < 0.01 versus CHIR (5 mM)-treated cultures. (D) Immunofluorescence staining of TBX18 and WT1 in D14 cultures treated with CHIR, RA/CHIR, or BMS493/ CHIR. Yellow in the inset panels indicates TBX18 and WT1 coexpression. Scale bars, 100 mm. Color images available online at www.liebertpub.com/scd

Article Snippet: Slides were then blocked with 5% goat or donkey serum in phosphate-buffered saline for 1 h and then incubated with primary antibodies against WT1 (diluted 1:1,000; ab89901; Abcam), TBX18 (diluted 1:100; sc17869; Santa Cruz), cTnT (0.5mg/mL; MAB1874; R&D), ZO1 (diluted 1:100; 339100; Life Science), CNN1 (diluted 1:10,000; C2687; Sigma), TAGLN (diluted 1:1,000; ab14106; Abcam), POSTN (diluted 1:1,000; ab14041; Abcam), or COL-1 (diluted 1:200; ab90395; Abcam) overnight at 4 C. Slides were then incubated with the relevant secondary antibody: Alexa Fluor 594 goat anti-rabbit IgG (111-585-003; Jackson), Alexa Fluor 488 goat anti-mouse IgG (115-545-003; Jackson), Alexa Fluor 488 donkey anti-goat IgG (705-545- 147; Jackson), Alexa Fluor 594 donkey anti-rabbit IgG (711- 585-152; Jackson), or Alexa Fluor 488-goat anti-rabbit IgG (111-545-003; Jackson) for 1 h at room temperature.

Techniques: Quantitative RT-PCR, Expressing, Gene Expression, Flow Cytometry, Imaging, Staining

Journal: Molecular medicine reports

Article Title: G9a inhibition promotes the formation of pacemaker-like cells by reducing the enrichment of H3K9me2 in the HCN4 promoter region.

doi: 10.3892/mmr.2022.12908

Figure Lengend Snippet: Figure 1. Characteristics of BMSCs and transfection efficiency. (A) Fluorescence-activated cell sorting analysis of CD29, CD44, CD34 and CD45 protein expression levels in BMSCs. (B) Cellular morphological features of BMSCs, which exhibited a spindle shape. Scale bar, 50 µm. (C) Red fluorescence of BMSCs after transfection with OE-Tbx18 for 48 h. Scale bar, 50 µm. (D) The microscopic morphology of BMSCs gradually evolved into strip-like features after 10 days of transduction by OE-Tbx18. Scale bar, 50 µm. (E) Western blotting of Tbx18 protein expression levels after transfection for 72 h. BMSCs without transfection were used as the control, BMSCs transfected with the empty vector were used as the NC. (F) Reverse transcription-quantitative PCR analysis of Tbx18 mRNA expression levels after transfection for 72 h. **P<0.01. BMSCs, bone marrow mesenchymal stem cells; OE-Tbx18, T-box 18 overex pression plasmid; NC, negative control.

Article Snippet: Primary antibodies against H3K9me2 (1:1,000; cat. no. 39239; Active Motif, Inc.), H3 (1:3,000; cat. no. H0164; Sigma-Aldrich; Merck KGaA), G9a (1:1,000; cat. no. sc-515726; Santa Cruz Biotechnology, Inc.), Nappa (1:1,000; cat. no. 13299-1-AP; ProteinTech Group, Inc.), Nappb (1:1,000; cat. no. 27426- 1-AP; ProteinTech Group, Inc.), Myh7 (1:1,000; cat. no. 22280-1-AP; ProteinTech Group, Inc.), β-actin (1:1,000; cat. no. SAB3500350; Sigma-Aldrich; Merck KGaA), HCN4 (1:1,000; cat. no. 55224-1-AP; ProteinTech Group, Inc.), cTnI (1:1,000; cat. no. 21652-1-AP; ProteinTech Group, Inc.), α-SA (1:1,000; cat. no. 23660-1-AP; ProteinTech Group, Inc.) and Tbx18 (1:1,000; cat. no. 23237-1-AP; ProteinTech

Techniques: Transfection, Fluorescence, FACS, Expressing, Stripping Membranes, Transduction, Western Blot, Control, Plasmid Preparation, Reverse Transcription, Real-time Polymerase Chain Reaction, Negative Control

Journal: Molecular medicine reports

Article Title: G9a inhibition promotes the formation of pacemaker-like cells by reducing the enrichment of H3K9me2 in the HCN4 promoter region.

doi: 10.3892/mmr.2022.12908

Figure Lengend Snippet: Figure 3. Expression of target protein after G9a inhibition treatment. (A) Western blotting demonstrated increased HCN4, α-SA and cTnI protein expression levels in different groups. (B) Quantitative analysis of the mRNA expression levels of HCN4, α-SA and cTnI. (C) Chromatin immunoprecipitation-qPCR analysis for H3K9me2 binding to the HCN4 promoter. The histogram showed the amount of immunoprecipitated DNA as detected using the qPCR assay. Values are indicated as % of input. *P<0.05 and **P<0.01. cTnI, cardiac troponin I; HCN4, hyperpolarization-activated cyclic nucleotide-gated channel 4; α-SA, α-striated actin; CM, cardiomyocyte; BIX, H3K9me2, histone H3 at lysine 9 dimethylation; Tbx18, T-box 18 overexpression plasmid.

Article Snippet: Primary antibodies against H3K9me2 (1:1,000; cat. no. 39239; Active Motif, Inc.), H3 (1:3,000; cat. no. H0164; Sigma-Aldrich; Merck KGaA), G9a (1:1,000; cat. no. sc-515726; Santa Cruz Biotechnology, Inc.), Nappa (1:1,000; cat. no. 13299-1-AP; ProteinTech Group, Inc.), Nappb (1:1,000; cat. no. 27426- 1-AP; ProteinTech Group, Inc.), Myh7 (1:1,000; cat. no. 22280-1-AP; ProteinTech Group, Inc.), β-actin (1:1,000; cat. no. SAB3500350; Sigma-Aldrich; Merck KGaA), HCN4 (1:1,000; cat. no. 55224-1-AP; ProteinTech Group, Inc.), cTnI (1:1,000; cat. no. 21652-1-AP; ProteinTech Group, Inc.), α-SA (1:1,000; cat. no. 23660-1-AP; ProteinTech Group, Inc.) and Tbx18 (1:1,000; cat. no. 23237-1-AP; ProteinTech

Techniques: Expressing, Inhibition, Western Blot, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Over Expression, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Improved Generation of Human Induced Pluripotent Stem Cell-Derived Cardiac Pacemaker Cells Using Novel Differentiation Protocols

doi: 10.3390/ijms23137318

Figure Lengend Snippet: Assessment of pacemaker-specific gene expression in cardiac pacemaker cells.

Article Snippet: TBX18 , T-box transcription factor 18 , Hs01385458_m1.

Techniques: Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Improved Generation of Human Induced Pluripotent Stem Cell-Derived Cardiac Pacemaker Cells Using Novel Differentiation Protocols

doi: 10.3390/ijms23137318

Figure Lengend Snippet: Overview of the used TaqMan probes and primers.

Article Snippet: TBX18 , T-box transcription factor 18 , Hs01385458_m1.

Techniques:

Journal: Adipocyte

Article Title: Characterization of the adiponectin promoter + Cre recombinase insertion in the Tg(Adipoq-cre)1Evdr mouse by targeted locus amplification and droplet digital PCR

doi: 10.1080/21623945.2020.1861728

Figure Lengend Snippet: Results of TLA Mapping of the Tg(Adipoq-cre)1Evdr mouse BAC insertion point. (a) TLA mapping of the amplicons of primer set 4 (red arrowhead) internal to Cre (orange text) confirmed the inserted BAC sequence for Adipoq promoter (blue bar and in blue text) included a Cre sequence that was located after the starting ATG of the inserted BAC (underlined in blue text). After the BAC start codon, there was one Cytosine residue (bolded in blue text) belonging to the BAC, followed by 18 inserted bases (black), and then two TG residues (underlined in orange text) of the start codon of Cre recombinase (GenBank sequence MK854762), so Cre is transcribed in frame. (b) TLA mapping of the amplicons of primer set 1 (red arrowhead) identified the transgene (blue bar and blue text) integration site on chromosome 9 within the intron connecting exons 6 and 7of the Tbx18 gene (red bar and red text), at a site with two homologous bases (purple text) found both in the normal genomic sequence and in the BAC. No recombination in the surrounding loci were detected

Article Snippet: Tbx18 qPCR Primers (ORIGENE, Rockville, MD, catalogue #MP216696) and beta-actin primers (Integrated DNA Technologies #88318771 and #88318794) were used in a final concentration of 0.5 μM in 1x Sybr Green Master Mix (Thermo Fisher, Waltham, MA, catalogue #4309155) with 100 ng cDNA.

Techniques: Sequencing

Journal: Adipocyte

Article Title: Characterization of the adiponectin promoter + Cre recombinase insertion in the Tg(Adipoq-cre)1Evdr mouse by targeted locus amplification and droplet digital PCR

doi: 10.1080/21623945.2020.1861728

Figure Lengend Snippet: Tbx18 gene expression in BL/6 and Tg(Adipoq-cre)1Evdr mice. Results of quantitative RT-PCR expressed as the relative mRNA expression of Tbx18 corrected for Beta-Actin. Epididymal white adipose tissue (eWAT) from mice hemizygous for Adipoq-Cre construct (yellow checkered bar) showed a 30% decrease in Tbx18 expression from eWAT from BL/6 mice (yellow bar) ( p = 0.0041). The hypothalamic expression in Tbx18 in Adipoq-Cre+ mice (black and white checkered bar) was not different from that of BL/6 mice (white bar)

Article Snippet: Tbx18 qPCR Primers (ORIGENE, Rockville, MD, catalogue #MP216696) and beta-actin primers (Integrated DNA Technologies #88318771 and #88318794) were used in a final concentration of 0.5 μM in 1x Sybr Green Master Mix (Thermo Fisher, Waltham, MA, catalogue #4309155) with 100 ng cDNA.

Techniques: Expressing, Quantitative RT-PCR, Construct