Proteome Profiler ヒト多能性幹細胞アレイキット Proteome Profiler Human Pluripotent Stem Cell Array Kit

siRNA-mediated Knockdown of PDGF R alpha, PDGF R beta, and cAbl Upregulates Expression of Pluripotent, Mesoderm, and Endoderm Markers. Small interfering RNAs (siRNAs) were transfected into mesenchymal stem cells by electroporation. Knockdown was verified by RT-PCR and Western blot analysis (data not shown here). Cell lysates from siRNA-treated cells were used in the Proteome Profiler Human Pluripotent Stem Cell Antibody Array (Catalog # ARY010) to examine relative expression levels of mesoderm, endoderm, and pluripotent markers. The images represent nitrocellulose membranes following chemiluminescent detection of bound analytes. Each of the array markers has duplicate spots, which are boxed to highlight their identity. A. Scrambled (Scr) siRNA-treated mesenchymal stem cells. B. Knockdown of PDGF R alpha upregulated pluripotent markers (Oct-3/4, Nanog, SOX2, and E-Cadherin) as well as markers of the mesoderm and endoderm compared to Scr. C. Knockdown of PDGF R beta upregulated expression of Snail, SOX17, VEGF R2, Oct-3/4, and Nanog compared to both Scr and PDGF R alpha knockdowns. D.Knockdown of cAbl upregulated pluripotent markers (Nanog, Oct-3/4, E-Cadherin, and SOX2) compared to Scr in addition to markers of the endoderm and mesoderm.

Adapted from Ball, S.G. et al. (2012) Stem Cells 30:548.

Abbreviations: AFP, alpha-Fetoprotein; GAPDH, Glyceraldehyde 3-Phosphate Dehydrogenase; GSC, Goosecoid; HCG, Human Chorionic Gonadotropin; HNF, Hepatocyte Nuclear Factor; PDGF R, Platelet-derived Growth Factor Receptor; Scr, scrambled; VEGF R, Vascular Endothelial Growth Factor Receptor.

Small Sample Lysate Volume Used to Detect Germ Layer Markers in Differentiated Human Pluripotent Stem Cells. BG01V human embryonic stem cells were differentiated into mesoderm, endoderm, and ectoderm using the differentiation supplements included in the Human Pluripotent Stem Cell Functional Identification Kit (Catalog # SC027B). Following differentiation in a 24-well plate, a single well of confluent cells was lysed in 100 µL of lysis buffer and analyzed using the Proteome Profiler Human Pluripotent Stem Cell Antibody Array (Catalog # ARY010). The differentiation supplements upregulated expression of the germ layer markers (A) Snail, (B) Sox17, and (C) Otx2 compared to undifferentiated cells. Immunocytochemistry for the same markers (Snail, Catalog # AF3639; Sox17, Catalog # AF1924; and Otx2, Catalog # AF1979; red) followed by staining with the appropriate NorthernLights™-conjugated Secondary Antibodies and counterstaining with DAPI (blue) is consistent with data obtained by array analysis.