Supplementary Figure 1: Addition of 2i rescues Prdm15^–/– phenotype.

(a) qPCR analysis of Prdm15 mRNA levels in Prdm15^{(+/+;+/–;–/–)} ESCs. (b) Western blot analysis of PRDM15 protein levels in Prdm15^{(+/+;+/–;–/–)} ESCs. TUBA is a loading control. (c) Analysis of PRDM15 protein (top) and RNA (bottom) levels in ESCs cultured in 2i versus SL. TBX3 is a naive pluripotency marker; uncropped gels are in Supplementary Data. In a and c, data are from three experiments; three independent ESC clones are shown per genotype. (d) Short-term growth analysis of Prdm15^{(+/+; +/–; –/–)} ESCs in 2i versus SL conditions. Cells (three independent cultures) were cultured in 2i for either 5 d (left) or switched to SL at day 3 (right); three independent clones are shown for each genotype. (e) Analysis of Prdm15 mRNA (top) and protein (bottom) levels in Prdm15^fl/fl versus Prdm15^Δ/Δ ESCs 3 d after OHT induction. (f) Colony formation assay in Prdm15^fl/fl versus Prdm15^Δ/Δ ESCs after 6 d of culture in SL. Data from two experiments with a total of n = 6 independent cell cultures are shown (g) Cell cycle analysis by flow cytometry; Prdm15^fl/fl versus Prdm15^Δ/Δ ESCs were pulsed with BrdU for 30 min and then stained with anti-BrdU/propidium iodide. (h) AP staining on Prdm15^fl/fl versus Prdm15^Δ/Δ ESCs cultured either in SL or 2i; data are presented as percent of the total number of colonies from three independent cultures. (h) Box plot of mean volumes for the teratomas formed by Prdm15^fl/fl versus Prdm15^Δ/Δ ESCs (n = 5 mice). Representative photos are shown to the right. Center values, mean; error bars, s.d. Student’s t test (two-sided) was used.