Cells were transduced with the LV-XEP-2Gi2R carrying the IRES-linked sequences for 2xGFP:NES and 2xRFP:NLS. We used the HIV-Rev NES (LQLPPLERLTL) and the SV40 Large-T NLS (PKKKRKV). For imaging, cells were transferred to tissue culture-treated ibidi μ-slides coated with astrocytes for iNs, PluriPro matrix (Cell Guidance Systems) for iPSCs, and uncoated for fibroblasts. Cell were fixed with 4% PFA for 20 min at room temperature and mounted in PVA-DABCO (Sigma Aldrich). Confocal images were taken on Zeiss LSM710 or LSM780 series confocal microscope. All data for one NCC experiment were acquired from cells cultured, transduced, and processed in parallel and images were taken on the same microscope with exactly the same settings reused. For analysis, 2 μm confocal sections through the nuclear layer were acquired from three confocal z stacks. Image J software was used to identify nuclear ROIs based on binarized DAPI channels and green and red fluorescence means were measured in the ROIs. Because of the morphological complexity of the astrocyte iN co-cultures, neuronal nuclear ROIs were selected manually. To quantify the integrity of cellular NCC, GFPnuc/RFPnuc were calculated in transgenic cells using Microsoft Excel and further processes in GraphPad Prism 6.
