Figure 5.
Regulation of ES cell survival by Bim. (A) Schematic of an inducible Bim ES cell line. Upon 4OH-T treatment, CreERT2 excises Neo selection marker and activates Bim transcription. (B) Western blots show that BimEL and BimL proteins are rapidly induced after 4OH-T treatment in two independent inducible ES cell lines. (C) Methylene blue stains show that most Bim-expressing cells are lost 2 d after 4OH-T induction. (D) Bim overexpression induces apoptosis in ES cells. Phase-contrast images and active caspase-3 stains are shown. (E) An activated Akt (myr-Akt) partially rescues cell growth defects of Ago1-4−/−; hAgo2Δ E7 cells after five passages with 4OH-T. Western blotting shows that HA-tagged myr-Akt is expressed in rescued E7 cells. Ago2-expressing E7 cells serve as the positive control for a complete rescue. (F) myr-Akt down-regulates Bim transcription in 4OH-T-treated E7 ES cells. Bim mRNA and protein levels are decreased in myr-Akt-rescued E7 cells. Shown are quantitative PCR and Western blot results. (G) The luciferase assay confirms loss of Ago2 and inability to use shRNA to silence a Ff-Luc reporter in myr-Akt-rescued cells. RT–PCR analysis shows that the majority of rescued E7 cells no longer express the floxed hAgo2 as a pool. The residual PCR signal from the pool might reflect a small population of rescued cells that still express hAgo2.