Figure 6. G1ER cells with high cyclin E activity recapitulate dyserythropoiesis phenotypes of cyclin ET74A T393A mice.
(A) Hbb-b1 transcript levels were measured in viable (non-propidium iodide retaining) G1ER cells transduced either with empty vector (MIGR1) or cyclin E, mutated at its N- and C-terminal phosphodegrons (cyclin E-AA), following induction with beta-estradiol for 48 hours. Inset - representative cell pellets are shown, made from undifferentiated G1ER cells (−E2) and cells transduced and differentiated as above. (B) Left panel - Bnip3L transcript levels were measured in the indicated, viable G1ER cell populations and are shown relative to expression in undifferentiated (−E2), empty vector-transduced (MIGR1) cells. Right panel – Lysates were prepared from differentiated G1ER cells and immunoblotted for the indicated proteins. (C) Mitochondrial superoxide levels were measured in live, G1ER cells with (+E2) and without (−E2) differentiation using Mitosox Red superoxide indicator. Inset shows increased poly-ADP-ribose polymerase (PARP) cleavage (* = 89 kD cleavage product, f.l. = full length PARP) in cyclin E-AA-transduced, differentiated G1ER cells (E-AA) and Annexin V-positive cells enumerated from two independent transductions.