mouse nkx2 5 antibody  (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: 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:

Journal: Frontiers in Cell and Developmental Biology

Article Title: Generation of NKX2.5 GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

doi: 10.3389/fcell.2021.797927

Figure Lengend Snippet: Establishment of NKX2.5 GFP hiPSC using CRISPR-Cas9. (A) Scheme of the strategy used for the generation of GFP KI at the NKX2.5 locus. (B) Schematic representation of the protocol used to induce CBiPS5 NKX2.5 GFP cells to cardiac lineages. (C) Brightfield and GFP images obtained from Clone 31 of CBiPS5 NKX2.5 GFP cells at day 17 of cardiac differentiation under in vivo fluorescence microscope. Scale bars, 200 μm. (D) Western blot of GFP and NKX2.5 proteins in CM derived from CBiPS5 NKX2.5 GFP and parental hiPSC line at day 20 of cardiac differentiation (left panel). Quantification of NKX2.5 protein level of both cell lines normalized to Tubulin is represented (right panel). (E) Dot plot diagrams of GFP + cells obtained by flow cytometry at days −4, 7, 8, 9, 10 and 11 of cardiac differentiation in the CBiPS5 NKX2.5 GFP and CBiPS5 parental line.

Article Snippet: One hundred thousand cells from NKX2.5 GFP -cFib cell cultures at passage 4 and parental non-differentiated CBiPS5 NKX2.5 GFP cells were immunostained with 100 ng of SSEA4-APC antibody (FAB1435A, R&D systems).

Techniques: CRISPR, In Vivo, Fluorescence, Microscopy, Western Blot, Derivative Assay, Flow Cytometry

Journal: Frontiers in Cell and Developmental Biology

Article Title: Generation of NKX2.5 GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

doi: 10.3389/fcell.2021.797927

Figure Lengend Snippet: NKX2.5-GFP + cell characterization. (A) Representation of the analyses performed in GFP + and GFP − cells sorted at day 11 of differentiation. (B) Dot plot diagram of GFP + cells obtained by flow cytometry analysis of pre-sorted CBiPS5 NKX2.5 GFP cells at day 11 of cardiac differentiation in two different assays. (C) Gene expression analysis of specific cardiac lineage markers in NKX2.5-GFP + (green circles) and NKX2.5-GFP - (black circles) cell derivatives at day 30 of cardiac differentiation. Two biological replicates (Exp1 and Exp2), three technical replicates in each. Median of three technical replicates in two biological replicates (Exp1 and Exp2) are represented; the p value is annotated in each graph, GFP − group vs GFP + group using Nested-t tests. (D) Activation frequency map for well #1 is shown. Baseline recordings acquired from 10 to 30 frames per second in a field of view of 2.5 mm (E) Activation frequency registrations (Hz) obtained by optical mapping of NKX2.5-GFP + cells-derived CM in three independent wells. (F) Patch-clamp recordings in GFP + cells-derived CM. Atrial and ventricular-like action potentials and percentages of each cardiomyocyte subtype are represented.

Article Snippet: One hundred thousand cells from NKX2.5 GFP -cFib cell cultures at passage 4 and parental non-differentiated CBiPS5 NKX2.5 GFP cells were immunostained with 100 ng of SSEA4-APC antibody (FAB1435A, R&D systems).

Techniques: Flow Cytometry, Expressing, Activation Assay, Derivative Assay, Patch Clamp

Journal: Frontiers in Cell and Developmental Biology

Article Title: Generation of NKX2.5 GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

doi: 10.3389/fcell.2021.797927

Figure Lengend Snippet: Characterization of NKX2.5 GFP -cFib. (A) Scheme of the fibroblast differentiation protocol used for the generation of NKX2.5 GFP -cFib derived from the CBiPS5 NKX2.5 GFP cell line. (B) Brightfield and GFP images obtained from CBiPS5 NKX2.5 GFP -cFib under in vivo fluorescence microscopy. Scale bars, 200 µm (C) Immunostaining analysis of fibronectin (in white) and collagen type I (in white) in NKX2.5 GFP -cFib. Nuclei: DAPI. Scale bars, 100 µm (D) Dot plot diagrams of SSEA4 + cells obtained by flow cytometry in CBiPS5 NKX2.5 GFP cells and NKX2.5 GFP -cFib. (E) Gene expression analysis of pluripotency (OCT4 and NANOG), cardiomyocyte (PLN and MYH6) and fibroblast (THY1, POSTN, VIM and TCF21) specific genes in NKX2.5 GFP -cFib, CBiPS5 NKX2.5 GFP cells and CBiPS5 NKX2.5 GFP -derived CM (NKX2.5 GFP -CM). (F) Gene expression analysis of fibroblast-specific markers (VIM, POSTN, TCF21 and THY1) in NKX2.5 GFP -cFib at passages 1, 2 and three and in patient-derived cFib, HFF-1 and HDF-α fibroblast cell lines. Mean ± SD represented of three technical replicates is represented.

Article Snippet: One hundred thousand cells from NKX2.5 GFP -cFib cell cultures at passage 4 and parental non-differentiated CBiPS5 NKX2.5 GFP cells were immunostained with 100 ng of SSEA4-APC antibody (FAB1435A, R&D systems).

Techniques: Derivative Assay, In Vivo, Fluorescence, Microscopy, Immunostaining, Flow Cytometry, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Generation of NKX2.5 GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

doi: 10.3389/fcell.2021.797927

Figure Lengend Snippet: Fibroblast activation assays in NKX2.5 GFP -cFib. (A) Immunostaining analysis of αSMA (in white) in NKX2.5 GFP -cFib non-treated and treated with 10 ng/ml TGFβ for 48 h. Nuclei: DAPI. Scale bars, 100 µm (B) Gene expression analysis of activated fibroblast markers (COL1A1, LOX, FN1, SMA) in NKX2.5 GFP -cFib and cFib derived from two different patients, non-treated (white bars) and treated (black bars) with 10 ng/ml TFGβ for 24 h. Three biological replicates were included in NKX2.5 GFP -cFib data, and three technical replicates in the case of patient-derived cFib. Mean ± S.D. of each data group are represented; non-treated vs treated p values are shown using a 2 way ANOVA test.

Article Snippet: One hundred thousand cells from NKX2.5 GFP -cFib cell cultures at passage 4 and parental non-differentiated CBiPS5 NKX2.5 GFP cells were immunostained with 100 ng of SSEA4-APC antibody (FAB1435A, R&D systems).

Techniques: Activation Assay, Immunostaining, Expressing, Derivative Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Generation of NKX2.5 GFP Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

doi: 10.3389/fcell.2021.797927

Figure Lengend Snippet: Potential applications of NKX2.5 GFP reporter cell lines in direct cardiac reprogramming.

Article Snippet: One hundred thousand cells from NKX2.5 GFP -cFib cell cultures at passage 4 and parental non-differentiated CBiPS5 NKX2.5 GFP cells were immunostained with 100 ng of SSEA4-APC antibody (FAB1435A, R&D systems).

Techniques: