TY - DATA T1 - Additional file 4: Figure S3. of Impaired osteogenesis in Menkes disease-derived induced pluripotent stem cells PY - 2015/09/07 AU - Dongkyu Kim AU - Jieun Choi AU - Kyu-Min Han AU - Beom Lee AU - Jin-Ho Choi AU - Han-Wook Yoo AU - Yong-Mahn Han UR - https://springernature.figshare.com/articles/figure/Additional_file_4_Figure_S3_of_Impaired_osteogenesis_in_Menkes_disease-derived_induced_pluripotent_stem_cells/4457384 DO - 10.6084/m9.figshare.c.3640997_D8.v1 L4 - https://ndownloader.figshare.com/files/7186682 KW - WT KW - MD 1-iPSCs KW - MD 1-fibroblasts KW - PCR KW - GAPDH KW - NESTIN KW - DAPI KW - GATA KW - EB KW - SMA KW - ATP 7A KW - TIFF 4321 kb KW - MD 2 patients KW - MD 2-fibroblasts KW - 500 Îź m KW - MD 2-iPSCs Strategy N2 - Characterization of MD-iPSCs. (A) Schematic defective regions of ATP7A in MD1 and MD2 patients. The functional domains of ATP7A are depicted. ATP7A has six copper-binding domains, one phosphatase domain, one phosphorylation domain, one ATP-binding domain, and eight transmembrane domains. The defective regions of each patient are marked as boxes. (B) Transcriptional expression of pluripotent genes in MD-fibroblasts and MD-iPSCs. GAPDH was used as a control. (C) In vitro differentiation of MD-iPSCs. Immunostaining of NESTIN (ectoderm, red), Îą-SMA (mesoderm, green), and GATA4 (endoderm, green) was performed 7 days after spontaneous EB differentiation. DAPI showed nuclear counterstaining (blue). Scale bar = 500 Îźm. (D) Karyotypes of MD1- and MD2-iPSCs. (E) Genetic mutations in MD1-fibroblasts and MD1-iPSCs. A single-base substitution was confirmed in MD1-fibroblasts and MD1-iPSCs (Figure E-a). The resultant PCR products were different in MD1-fibroblasts and MD1-iPSCs (Figure E-b). The size difference (450 bp in WT and 246 bp in MD1) generated by exon skipping was analyzed by gel electrophoresis. (F) Mutations in MD2-fibroblasts and MD2-iPSCs. Strategy for duplex PCR was explained as an illustration (Figure F-a). Size differences (532 bp in WT and 224 bp in MD2) generated by a large genomic deletion between WT- and MD2-fibroblasts were detected (Figure F-b). The primers used in this study are listed in Additional file 1 (Table S2). (TIFF 4321 kb) ER -