Human NKX2.5 Antibody

Detection of Human NKX2.5 by Western Blot

EOMES programs hESCs into functional CMs at high efficiency. a Illustration of growth factor-mediated and optimized EOMES induction-based cardiac differentiation protocols. Bottom right: Immunoblot validating doxycycline-dependent EOMES expression in a transgenic EOMESKO/E.TET-ON hESC line. b Typical yields of hESC-CMs (left, flow cytometry) and NKX2.5 expression (right, immunoblot) obtained with the two protocols (day 10). c Immunostainings 21 days after the initiation of EOMES induction. Weak perinuclear ANP staining is typical in overall MLC2v-positive hESC-CMs. Scale bars: 25 (top) and 50 µm (bottom). d Acceleration and slowdown of spontaneous beat rates in pCMs following exposure to 10 µM isoprenaline and 10 µM propranolol, respectively, on multielectrode arrays. e Microarray-based time course analysis comparing the indicated protocols and cell lines. RESCUE cells carry an inducible EOMES transgene on EOMESKO HuES6 background. Underlying data are from Supplementary Data 2 Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29382828), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human NKX2.5 by Immunocytochemistry/Immunofluorescence

Contextual requirements of EOMES-mediated CM programming. a DOX dose dependency of the EOMES TET-ON protocol using the WTE.TET-ON hESC line. Top panel: Immunostains at 1.5 wk. Scale bar: 100 µm. Bottom: Flow cytometry analysis. Numbers indicate average percentages of cTnT-positive CMs from 3–6 experiments per condition. b CM programming by EOMES necessitates suppression of autocrine WNT signaling from the third day of transgene induction (qPCR data, n = 2). c DOX dose-dependent CM programming using an independent WTE.TET-ON hiPSC line. The data shows immunostaining (scale bar: 100 µm), RT-qPCR (n = 4), and FACS analysis (n = 3) performed at 1 wk of differentiation. The asymmetrical shape of the DOX titration data with this line is in part due to the incomplete repression of SOX2 at 0.05 µg/ml, which caused overgrowth of the cultures with neural precursors (also see Supplementary Fig. 3f). Error bars: s.e.m. Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29382828), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Human NKX2.5 by Immunocytochemistry/Immunofluorescence

EOMES knockout hESCs fail to differentiate into cardiomyocytes. a Immunoblot confirming EOMES expression and its absence in WT and KO cells, respectively, at the cardiac mesoderm stage of directed differentiation. b EOMES KO cells fail to express the early cardiomyocyte marker NKX2.5 following exposure to a directed differentiation protocol. Scale bar: 50 µm. c EOMES KO cells show a general failure in markedly upregulating essential pan-cardiac genes (qPCR data, n = 3; error bars: s.e.m.). For each gene, the expression level in the highest expressing sample (“max” = WT in all three cases) is set to 1. d Differentiation of EOMES KO hESCs under signaling factor-assisted, non-cardiac mesoderm/endoderm-permissive culture conditions. Top: EOMES KO cells fail to express alpha-fetoprotein following Activin A-assisted endodermal induction. Scale bar: 50 µm. Bottom: Non-cardiac mesodermal differentiation competence of EOMES KO hESCs. KO cells were differentiated using varied meso and endoderm-permissive induction conditions. The data denote transformed p-values of annotation terms enriched in gene sets upregulated in EOMES KO samples compared to undifferentiated hESCs or differentiated WT controls (cutoffs: 10 or 3-fold, respectively). Underlying data: Supplementary Data 1 Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/29382828), licensed under a CC-BY license. Not internally tested by R&D Systems.