Additional file 1: Figure S1. of CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons

Analysis of premutation iPSCs and full mutation ESCs. a Immunostaining for pluripotency markers in SC120 and HT14 iPSCs and WCMC37 ESCs. Cells were grown in 24-well plates, fixed and stained for the indicated pluripotency markers (red) as described in the supplemental experimental procedure. Nuclei were stained with DAPI (blue). Scale bar: 200 μm. b SC120 iPSCs were differentiated into neurons and stained for neuronal markers. SC120 neural progenitor cells (NPC) were stained at passage 2 with Nestin (green) and Sox1 (red). Scale bar: 200 μm. SC120 neurons were stained at 4 weeks of differentiation with Map2 (red) and TuJ1 (red). Nuclei were stained with DAPI (blue). Scale bar: 100 μm. c DNA methylation at the FMR1 promoter was analyzed by qMS-PCR in SC120 iPSCs, NPCs, and neurons at indicated weeks (wk) of differentiation. d Immunostaining for FMRP (red) was done in indicated cell lines. The percentage of FMRP-positive cells is indicated under each panel. In SC120 iPSCs, all the cells were positive for FMRP expression but the intensity was much reduced compared to the control H1 cells. Scale bar: 100 μm. e Western blot for FMRP levels in indicated cell lines. β-Actin is used as loading control. f Western blot analysis for MSH2, MSH3, and MSH6 proteins in stem cells and fibroblasts was done as described in the supplemental experimental procedures. β-Actin is used as loading control. (TIFF 11721 kb