Additional file 4: Figure S3. of Impaired osteogenesis in Menkes disease-derived induced pluripotent stem cells

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)