nkx2 5 rabbit igg santa cruz biotech sc14033  (Santa Cruz Biotechnology)

Journal: Communications Biology

Article Title: Computational profiling of hiPSC-derived heart organoids reveals chamber defects associated with NKX2-5 deficiency

doi: 10.1038/s42003-022-03346-4

Figure Lengend Snippet: a Diagram of the strategy to generate an isogenic line carrying an EA-associated mutation (PM28, PM52) and a control line with a large deletion on Nkx2-5 (Del33). b Representative organoids differentiated from the two mutants at day 30. c Nkx2-5 expression significantly reduced in the Del33 line derived organoids. Scale bar = 100 μm. d The percentages of beating and non-beating organoids in the control and mutant at RA- and RA+ groups. n = 3 biological independent organoid differentiation. e The beating rates of control and mutant heart organoids. n = 20 organoids in controls, n = 20 and 10 organoids in Del33 RA+ and A-, n = 10 and 11 organoids in PM28 RA+ and A-, respectively. *** p < 0.001, **** p < 0.0001, Student’s t test against WTC. f (i) Representative plots of Ca 2 + transients in mutant and control organoid CMs at day 30. (ii) Quantification of the transient duration 50, transient duration 90 and time to 50% decay in WT and mutant organoid CMs. n = 81 cells in WT RA-, n = 208 cells in WT RA+, n = 77 cells in Del33 RA-. **** p < 0.0001, Student’s t test against WT RA-. g High expression of atrial genes and low expression of ventricular genes in RA- mutant CMs. Scale bar = 100 μm. h Defective sarcomere structures were identified in mutant heart organoids and sarcomere length was quantified based on the ACTN2-eGFP signal. n = 23 sarcomeres in each group, data was displayed as mean ± SEM. **** p < 0.0001, Student’s t test against WTC. Scale bar = 20 μm.

Article Snippet: The mouse anti-Cardiac Troponin T (5 µg/ml, Invitrogen, MA5-12960), mouse anti-human COUP-TF II/NR2F2 antibody (1:1000, R&D Systems, PP-H7147-00), rabbit anti-MYL7 antibody (1:1000, Sigma, SAB2701294), rabbit anti-Cardiac Troponin T (1:400, Abcom, ab45932), mouse anti- MYH6 (1:50, DSHB, S46), mouse anti- MYH7 (1:50, DSHB, BA-D5), mouse anti- ID2 (1:50, DSHB, PCRP-ID2-1A8), mouse anti- HEY2 (1:50, DSHB, PCRP-HEY2-1H10), mouse anti- COL1A1 (1:50, DSHB, SP1.D8), mouse anti-NFATC1 (1:25, DSHB, PCRP-NFATC1-1A2), rabbit anti-VE-Cadherin (1:400, Cell signaling, #2500), mouse anti-Nkx2-5 (25 μg/ml, R&D Systems, # MAB2444), rabbit anti-Myosin, Smooth Muscle Heavy Chain (1;200, Biomedical Technologies, BT-562), mouse anti-MF20 (1:100, DSHB, MF 20) were used.

Techniques: Mutagenesis, Expressing, Derivative Assay

Journal: Clinical Epigenetics

Article Title: Identification of miR-20b-5p as an inhibitory regulator in cardiac differentiation via TET2 and DNA hydroxymethylation

doi: 10.1186/s13148-024-01653-7

Figure Lengend Snippet: Establishment of hESCs-derived in vitro cardiac differentiation. A Scheme of in vitro cardiac differentiation derived from hESCs. B Immunofluorescence of cTnT after 12-day differentiation (Scale bar = 20 μm). C-G The relative mRNA expression levels of cardiac markers ( GATA4 , NKX2.5 , TBX5 , MYH6 and cTnT) during 12-day differentiation were tested by qRT-PCR ( n = 3-4). H The relative protein levels of cardiac markers (GATA4, NKX2.5, TBX5, MYH6 and cTnT) during 12-day differentiation were detected by western blotting ( n = 3). I Flow cytometry analysis of cTnT-positive cell during 12-day differentiation ( n = 3). D0, D3, D6, D9 and D12 indicated the time points at Day 0, Day 3, Day 6, Day 9 and Day 12, respectively. Quantitative data were presented as mean ± SEM, while statistical significance was analyzed via a one-way ANOVA followed by Bonferroni multiple comparisons test and represented as * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: To block non-specific binding, the membranes were incubated in 5% non-fat milk for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies including rabbit anti-α-tubulin (diluted 1:1000, Abcam, ab52866), rabbit anti-GAPDH (diluted 1:1000, Cell Signaling Technology, #5174), rabbit anti-TET2 (diluted 1:1000, Cell Signaling Technology, #45010), rabbit anti-TBX5 (diluted 1:1000, Abcam, ab137833), mouse anti-NKX2-5 (diluted 1:1000, Abcam, ab91196), rabbit anti-GATA4 (diluted 1:1000, Abcam, ab134057), rabbit anti-cTnT (diluted 1:1000, Abcam, ab209813) and mouse anti-MYH6 (diluted 1:1000, Abcam, ab50967).

Techniques: Derivative Assay, In Vitro, Immunofluorescence, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry

Journal: Clinical Epigenetics

Article Title: Identification of miR-20b-5p as an inhibitory regulator in cardiac differentiation via TET2 and DNA hydroxymethylation

doi: 10.1186/s13148-024-01653-7

Figure Lengend Snippet: Inhibition of hESC-derived cardiac differentiation by miR-20b-5p. A-C qRT-PCR analysis of miR-20b-5p level during 12-day cardiac differentiation in hESCs-CMs A , hiPSCs-CMs B and mESCs-CMs C ( n = 3–4). D qRT-PCR analysis of miR-20b-5p level in embryonic and neonatal heart samples (E17.5, P2 and P9) from wild-type C57BL/6 mice ( n = 3). E qRT-PCR analysis of the relative mRNA expression level of cardiac transcriptional factors ( GATA4 , NKX2.5 , TBX5 , MYH6 and cTnT ) in 12-day cardiac-differentiated hESCs with treatment of m-NC (mimic-negative control), miR-20b-5p-mimic, i-NC (inhibitor-negative control) or miR-20b-5p-inhibitor ( n = 3–5). F and G Western blot analysis G and quantification F of the relative protein levels of cardiac transcriptional factors (GATA4, NKX2.5, TBX5, MYH6 and cTnT) in 12-day cardiac-differentiated hESCs with treatment of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor ( n = 3). H Immunofluorescence of cTnT in 12-day cardiac-differentiated hESCs with treatment of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (Scale bar = 10 μm). I and J Flow cytometry analysis I and quantification J of cTnT-positive cells in 12-day cardiac-differentiated hESCs with treatment of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor ( n = 3). D0, D3, D6, D9 and D12 indicated the time points at Day 0, Day 3, Day 6, Day 9 and Day 12, while E17.5, P2 and P9 indicated Embryonic day 17.5, Postnatal day 2 and 9, respectively. Quantitative data were presented as mean ± SEM, while statistical significance was analyzed via a one-way ANOVA followed by Bonferroni multiple comparisons test and represented as * P < 0.05, ** P < 0.01 and *** P < 0.001

Article Snippet: To block non-specific binding, the membranes were incubated in 5% non-fat milk for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies including rabbit anti-α-tubulin (diluted 1:1000, Abcam, ab52866), rabbit anti-GAPDH (diluted 1:1000, Cell Signaling Technology, #5174), rabbit anti-TET2 (diluted 1:1000, Cell Signaling Technology, #45010), rabbit anti-TBX5 (diluted 1:1000, Abcam, ab137833), mouse anti-NKX2-5 (diluted 1:1000, Abcam, ab91196), rabbit anti-GATA4 (diluted 1:1000, Abcam, ab134057), rabbit anti-cTnT (diluted 1:1000, Abcam, ab209813) and mouse anti-MYH6 (diluted 1:1000, Abcam, ab50967).

Techniques: Inhibition, Derivative Assay, Quantitative RT-PCR, Expressing, Negative Control, Western Blot, Immunofluorescence, Flow Cytometry

Journal: Clinical Epigenetics

Article Title: Identification of miR-20b-5p as an inhibitory regulator in cardiac differentiation via TET2 and DNA hydroxymethylation

doi: 10.1186/s13148-024-01653-7

Figure Lengend Snippet: Reversal of TET2 knockdown-mediated suppression in hESCs-derived cardiac differentiation by inhibiting miR-20b-5p. A qRT-PCR analysis of the relative mRNA expressions levels of TET2 and cardiac transcriptional factors ( GATA4 , NKX2.5 , TBX5 , MYH6 and cTnT ) in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of m-NC (mimic-negative control) or miR-20b-5p-mimic ( n = 3). B qRT-PCR analysis of the relative mRNA expressions levels of TET2 and cardiac transcriptional factors ( GATA4 , NKX2.5 , TBX5 , MYH6 and cTnT ) in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of i-NC (inhibitor-negative control) or miR-20b-5p- inhibitor ( n = 3). C and E Western blot analysis C and quantification E of the relative protein levels of TET2 and cardiac transcriptional factors (GATA4, NKX2.5, TBX5, MYH6 and cTnT) in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of m-NC or miR-20b-5p-mimic ( n = 3). D and F Western blot analysis D and quantification F of the relative protein levels of TET2 and cardiac transcriptional factors (GATA4, NKX2.5, TBX5, MYH6 and cTnT) in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of i-NC or miR-20b-5p-inhibitor ( n = 3). G-I Flow cytometry analysis G and quantification H and I of cTnT-positive cell in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor ( n = 3). J and K Immunofluorescence staining of TET2 J and cTnT K in 12-day cardiac-differentiated hESCs with TET2 knockdown and transfection of m-NC, miR-20b-5p-mimic, i-NC or miR-20b-5p-inhibitor (Scale bar = 10 μm). Quantitative data were presented as mean ± SEM. Statistical significance was analyzed via a one-way ANOVA followed by Bonferroni multiple comparisons test and represented as * P < 0.05, ** P < 0.01 and *** P < 0.001, while n.s. indicated non-significance

Article Snippet: To block non-specific binding, the membranes were incubated in 5% non-fat milk for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with primary antibodies including rabbit anti-α-tubulin (diluted 1:1000, Abcam, ab52866), rabbit anti-GAPDH (diluted 1:1000, Cell Signaling Technology, #5174), rabbit anti-TET2 (diluted 1:1000, Cell Signaling Technology, #45010), rabbit anti-TBX5 (diluted 1:1000, Abcam, ab137833), mouse anti-NKX2-5 (diluted 1:1000, Abcam, ab91196), rabbit anti-GATA4 (diluted 1:1000, Abcam, ab134057), rabbit anti-cTnT (diluted 1:1000, Abcam, ab209813) and mouse anti-MYH6 (diluted 1:1000, Abcam, ab50967).

Techniques: Derivative Assay, Quantitative RT-PCR, Transfection, Negative Control, Western Blot, Flow Cytometry, Immunofluorescence, Staining

Journal: Reports of Biochemistry & Molecular Biology

Article Title: Effect of miR-18a-5p, miR-19a-3p, and miR-20a-5p on In Vitro Cardiomyocyte Differentiation of Human Endometrium Tissue-Derived Stem Cells Through Regulation of Smad4 Expression

doi: 10.52547/rbmb.12.1.136

Figure Lengend Snippet: Primer sequences used for qRT-PCR.

Article Snippet: The cells were then incubated with mouse monoclonal primary antibodies against human Nkx2-5 (Biorbyt, UK) Smad4 (Santa Cruz Biotechnology Inc., USA) and cTnT (Biorbyt, UK) for 24 hours at 2-8 oC.

Techniques: