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: Stem Cells International

Article Title: Genetically Modified Porcine Mesenchymal Stem Cells by Lentiviral Tbx18 Create a Biological Pacemaker

doi: 10.1155/2019/3621314

Figure Lengend Snippet: Primer sequences used in the study.

Article Snippet: Primary antibodies against HCN4 (ab32675, 1 : 5000), cTnI (ab47003, 1 : 500), α -SA (ab156302, 1 : 1000), Tbx18 (ab115262, 1 : 800), and GAPDH (Proteintech, 1 : 5000) were incubated overnight at 4°C.

Techniques:

Journal: Stem Cells International

Article Title: Genetically Modified Porcine Mesenchymal Stem Cells by Lentiviral Tbx18 Create a Biological Pacemaker

doi: 10.1155/2019/3621314

Figure Lengend Snippet: Cell characteristics of BMSCs and demonstration of gene transfer. (a) Surface marker of BMSCs under flow cytometry. BMSCs: bone mesenchymal stem cells; CD: cluster of differentiation. (b) Cellular morphological feature of BMSCs exhibiting a spindle shape. Scale bar = 400 μ m. (c) Green fluorescence of BMSCs after transfection with Lenti-Tbx18 for 48 h. Scale bar = 400 μ m. (d, e) Representative morphological characterizations of BMSCs with a strip shape and original spindle shape after transduction with Lenti-Tbx18 and Lenti-GFP for 7 days, respectively. Scale bar = 200 μ m.

Article Snippet: Primary antibodies against HCN4 (ab32675, 1 : 5000), cTnI (ab47003, 1 : 500), α -SA (ab156302, 1 : 1000), Tbx18 (ab115262, 1 : 800), and GAPDH (Proteintech, 1 : 5000) were incubated overnight at 4°C.

Techniques: Marker, Flow Cytometry, Fluorescence, Transfection, Stripping Membranes, Transduction

Journal: Stem Cells International

Article Title: Genetically Modified Porcine Mesenchymal Stem Cells by Lentiviral Tbx18 Create a Biological Pacemaker

doi: 10.1155/2019/3621314

Figure Lengend Snippet: Western blotting and PCR analysis of target protein and mRNA expression level after transfection. (a) Western blotting of Tbx18 expression level after transfection for 72 h. BMSCs without transfection are set as the control. (b) PCR analysis of Tbx18 expression level after transfection for 48 h. ∗ represents P < 0.05. (c) Western blotting detects increased HCN4, α -SA, and cTnI protein expression. (d) Quantitative analysis of the protein expression levels of HCN4, α -SA, and cTnI. ∗ represents P < 0.05. cTnI: cardiac troponin I; GFP: green fluorescent protein; HCN4: hyperpolarization-activated cyclic nucleotide-gated channel 4; α -SA: α -striated actin.

Article Snippet: Primary antibodies against HCN4 (ab32675, 1 : 5000), cTnI (ab47003, 1 : 500), α -SA (ab156302, 1 : 1000), Tbx18 (ab115262, 1 : 800), and GAPDH (Proteintech, 1 : 5000) were incubated overnight at 4°C.

Techniques: Western Blot, Expressing, Transfection, Control

Journal: Stem Cells International

Article Title: Genetically Modified Porcine Mesenchymal Stem Cells by Lentiviral Tbx18 Create a Biological Pacemaker

doi: 10.1155/2019/3621314

Figure Lengend Snippet: BMSCs-Tbx18 create a biological pacemaker. (a) Photograph of the implantation procedure for transduced cells. Yellow arrow represents the pacing lead of the temporary epicardial pacemaker, and white arrow represents the implantation site of the right ventricle. (b) Mean heart rate of the BMSC-Tbx18 group and BMSC-GFP group after injection for 2 days. Data are means ± SEM. ∗ P < 0.05 compared to the BMSC-GFP group. (c) Mean heart rate of both groups after injection for continuous 5 weeks. Data are means ± SEM. ∗ P < 0.05 compared to the BMSC-GFP group. (d) Increasement in heart rate after continuous isoproterenol infusion for 10 min between both two animal groups. ∗ P < 0.05 compared to the BMSC-GFP group.

Article Snippet: Primary antibodies against HCN4 (ab32675, 1 : 5000), cTnI (ab47003, 1 : 500), α -SA (ab156302, 1 : 1000), Tbx18 (ab115262, 1 : 800), and GAPDH (Proteintech, 1 : 5000) were incubated overnight at 4°C.

Techniques: Injection

Journal: Stem Cells International

Article Title: Genetically Modified Porcine Mesenchymal Stem Cells by Lentiviral Tbx18 Create a Biological Pacemaker

doi: 10.1155/2019/3621314

Figure Lengend Snippet: BMSC-Tbx18 biological pacemaker activity originates from the right ventricle. (a–c) Representative ECG of the temporary epicardial pacemaker, BMSC-Tbx18 group, and BMSC-GFP group; the pacing rate of the epicardial pacemaker was preset at 80 bpm as a control. (d–f) Enlarged figure of corresponding ECG for the temporary epicardial pacemaker, BMSC-Tbx18 group, and BMSC-GFP group; red arrows represent the prolonged QRS complex (duration > 0.12 s), and black arrow represents the ventricular pacer spike.

Article Snippet: Primary antibodies against HCN4 (ab32675, 1 : 5000), cTnI (ab47003, 1 : 500), α -SA (ab156302, 1 : 1000), Tbx18 (ab115262, 1 : 800), and GAPDH (Proteintech, 1 : 5000) were incubated overnight at 4°C.

Techniques: Activity Assay, Control

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