Fig. 1. Ring1A/B are required for the maintenance of mouse ES cell identity. (A) Western blot analysis showing the kinetics of Ring1B depletion at 0, 3, 6, 12, 24 and 48 hours after treatment of Ring1A^-/-;Ring1B^fl/fl;Rosa26::CreERT2 ES cells with 4-hydroxy tamoxifen (OHT). Lamin B served as a loading control. (B) Western blot showing Ring1B and mono-ubiquitylated H2A (H2Aub1) depletion 2 days after treatment with OHT in Ring1A^-/-;Ring1B^fl/fl;Rosa26::CreERT2 ES cells. OHT was present in (+) or absent from (-) the ES cell culture medium. Coomassie Brilliant Blue (CBB) staining for histones was used as a loading control. (C) Morphology of conditional Ring1A/B-dKO ES cells. Ring1A^-/-;Ring1B^fl/fl;Rosa26::CreERT2 ES cells were cultured in the absence (-OHT) or presence (+OHT) of OHT, which represent the single Ring1A-KO or Ring1A/B-dKO cells, respectively. At day 2, Ring1A/B-dKO ES cells retain ES-cell-like morphology; however, from day 3-4, Ring1A/B-dKO ES cells begin to lose ES-cell-like morphology. (D) Gene ontology (GO) analysis of genes more than 2-fold derepressed 4 days after OHT treatment of Ring1A^-/-;Ring1B^fl/fl;Rosa26::CreERT2 ES cells. The significance (P-value) of the enrichment of each GO term is indicated for each category of biological process. For details, see Table S1 in the supplementary material. (E) Changes in expression levels of Hoxa9, Hoxb4, Hoxb8, Gata6, Cdx2, Zic1 and T at 2 and 4 days after OHT treatment (+OHT) of Ring1B^fl/fl;Rosa26::CreERT2 or Ring1A^-/-;Ring1B^fl/fl;Rosa26::CreERT2 ES cells as determined by real-time PCR. Expression levels were normalized to an Actb control and are depicted as fold changes relative to the OHT-untreated (-OHT) ES cells. Error bars are based on the s.d. as derived from triplicate PCR reactions. (F) Ring1A^-/-;Ring1B^fl/fl;Rosa26::CreERT2 ES cells were cultured in the absence (-OHT, upper panels) or presence (+OHT, day 4, lower panels) of OHT, and were immunostained with antibodies to Oct3/4 (green) and Gata4 (red). The left-most panels show nuclei stained with Hoechst 33342 (blue); the right-most panels show merged images. Arrowheads indicate differentiated cells that express Gata4 but not Oct3/4. Arrows indicate feeder cells. Scale bars: 200 µm in C; 45 µm in F.