Fig. 6.
YY1 REPO domain/PcG function is crucial for early T cell survival. (A) Experimental strategy: Yy1f/f Vav-Cre fetuses were harvested on E14.5. Lin− fetal liver cells were transduced with MigR1, MigR1-FlagYY1 or MigR1-FlagYY1ΔREPO. Yy1f/f Lin− fetal liver cells were transduced with MigR1 vector. Retrovirally infected Lin− fetal liver cells were co-cultured with OP9-DL1 feeder cells in the presence of IL7 and Flt-3L. Cells were harvested for flow analysis on day 3 and day 10. (B) Flow cytometry gating of DN T cells at day 3 of co-culturing. (C) Absolute number of Lin− fetal liver cells in Yy1−/−+MigR1, Yy1−/−+YY1, Yy1−/−+YY1ΔREPO and Yy1+/++MigR1 groups before plating. (D) Percentage of GFP+ DN1 (Lin−CD44+CD25−) cells at day 3 of co-culturing. (E) Absolute number of GFP+ DN1 cells at day 3 of co-culturing. (F) Representative flow gating strategy for early apoptosis (annexin V+DAPI−), late apoptosis (annexin V+DAPI+) and non-apoptotic cells (annexin V−DAPI−). (G) Quantification of non-apoptotic, early apoptotic and late apoptotic DN1 cells at day 3 of co-culturing. (H) Representative gating strategy for Ki67/DAPI cell proliferation assay at day 10 of co-culturing. Cells in the G0 phase were defined as Ki67−DAPI−. Cells in the G1 phase were defined as Ki67+DAPI−. Cells in S/G2/M phase were defined as K67+DAPI+. (I) Quantification of DN1 cells in G0, G1 and S/G2/M phases at day 10 of co-culture. n represents the number of individual fetal livers. Experiments were repeated four times in C-E and twice in G and I; panels C,D and E were analyzed by one-way ANOVA; panels G and I were analyzed by two-way ANOVA. Data are presented as mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.