Extended Data Figure 4: Confirmation of CAF-1 reprogramming phenotype with alternative transgenic and non-transgenic vector systems.

a, Alkaline phosphatase (AP)-positive, transgene-independent iPS cell colonies at day 14 following transduction of R26-M2rtTA MEFs with tetO-STEMCCA lentiviral OKSM expression vector and either Chaf1a.164 or Ren.713 shRNA vectors and treatment with high (2 μg ml⁻¹) or low (0.2 μg ml⁻¹) doses of doxycycline for 10 days. b, Quantification of data shown in a. Experiment was performed at 3 different plating densities (n = 1 experiment per density), representative data are shown. c, Comparison of reprogramming efficiencies between Col1a1::tetOP-OKSM; R26-M2rtTA reprogrammable MEFs and wild-type MEFs infected directly with OKSM-expressing lentiviral vectors containing either a strong Ef1a full-length promoter (Ef1a-OKSM long) or a weaker truncated promoter (Ef1a-OKSM short). TRE3G-OKSM is a lentiviral vector with a strong promoter, whose activity is downregulated over time upon infection of CAGS-rtTA3 transgenic MEFs (see below). Error bars show s.d. from biological triplicates. d, Quantitative RT–PCR data showing variability in OKSM expression levels over time using different vector systems. Cells were analysed after 3 and 6 days of infection (lentiviral vectors) or doxycycline exposure (reprogrammable MEFs). Error bars show s.d. from biological triplicates. OGR MEF, transgenic MEFs carrying Oct4–GFP and CAGS-rtTA3 alleles. e, Quantification of Oct4 protein levels by intracellular flow cytometry (top) and cellular granularity/complexity by side scatter (SSC) analysis of indicated samples (bottom). Error bars show s.d. from biological triplicates.