Figure 3.
XCR during Active Reprogramming
(A) Crystal violet staining of a reprogrammed whole cell in a six-well plate. 6TG-resistant fLNS-6TG (XaHPRT−XiHPRT+) cells were induced for reprogramming in a normoxic or hypoxic condition using pMIG retroviral vector, or in a normoxic condition using STEMCCA lentiviral vector. After 10 days, reprogramming was continued in medium with HAT or 6TG for 2 weeks. Seven days after HAT was withdrawn, the plate was stained for crystal violet. In all three conditions, iPSCs with resistance to HAT were formed.
(B) Allelic-specific sequencing of GYG2 and MAOA in cells under 14 days of reprogramming.
(C) Immunostaining of wild-type (WT) MECP2 in dfRTT3-46m cells undergoing reprogramming at the indicated times with antibody recognizing the C terminus of MECP2 (red) and DAPI (blue). White arrows indicate WT of MECP2 and yellow arrows indicate the mutant type of MECP2. Scale bar, 20 μm.
(D) Quantification of WT MECP2+ cells in dfRTT3-46m cells undergoing reprogramming in (C). Four randomly chosen fields were used to count the number of MECP2+ cells and to calculate the percentage. Error bars represent mean ± SEM.
(E) Protein expression of WT of MECP2 in dfRTT3-46m fibroblasts undergoing reprogramming. At 10 and 21 days after reprogramming, whole-cell extracts were immunoblotted for MECP2 antibody recognizing the C terminus of MECP2.
(F) Representative SNP of MECP2 in secondary iPSCs.