Fig. 5. p57^KIP2 is essential to CFUe self-renewal in vitro.

(A) p57^KIP2 mRNA levels during CFUe self-renewal in vitro and following the switch to differentiation. Wild-type S0 cells were cultured for 5 days in self-renewal medium (“Dex + Epo”). Cells were washed and then placed either in differentiation medium (“Epo”) or back in self-renewal medium (“Epo + Dex”). Dex withdrawal leads to rapid down-regulation of p57^KIP2 and to concurrent rapid induction of erythroid genes, such as α-globin or β-globin. mRNA measured by qRT-PCR, normalized to β-actin, and expressed relative to t = 0. (B) Western blot and protein band quantification during CFUe self-renewal in vitro and following the switch to differentiation. Experiment as in (A); on day 0, CFUe progenitors were replaced either in self-renewal medium (Epo + Dex) or in differentiation medium (Epo). The p57^KIP2 band was identified using control cells transduced with retroviral vector expressing p57^KIP2, as in (E). The uppermost band on the p57^KIP2 blot is an unrelated cross-reacting band seen in Epo + Dex cultures [see (E)]. Legend as in (A), except that blue diamonds and red circles represent α-globin. (C) Cell cycle status of CFUe during self-renewal in vitro (Epo + Dex) and 20 to 60 hours following Dex withdrawal (Epo). BrdU MFI in Epo is expressed relative to its value in matched control cells undergoing Dex-dependent self-renewal. Top: Representative example. Bottom: Summary of seven matched cultures from three independent experiments. Statistical significance, paired t test. (D) p57^KIP2-deficient S0 CFUe cells fail to self-renew in vitro. S0 cells derived from individual wild-type or littermate p57^KIP2-deficient fetal livers were cultured in medium containing Epo + Dex. Cell numbers are relative to t = 0, mean ± SE of four (for +/−m) or five (for −/−) embryos. See also fig. S4A. (E) Western blot of p57^KIP2 protein on day 9 of Dex-dependent CFUe self-renewal in vitro. Control 3T3 cells transduced with either empty vector or vectors expressing each of two p57^KIP2 isoforms are also shown. Fetal liver cells express only the shorter 335–amino acid isoform of p57^KIP2. Low levels of the p57^KIP2 protein are also detectable in p57^KIP2+/−m cells following 9 days of culture in Epo + Dex (see also fig. S4, B and C). Note that the top band is an unrelated cross-reacting band. (F) Increased intra–S-phase DNA synthesis rate in p57^KIP2-deficient CFUe undergoing Dex-dependent self-renewal in vitro for 6 days. BrdU MFI in the S-phase gate is expressed relative to the wild-type littermate value. Representative of three independent experiments. (G) Increased number of γH2AX-positive cells in p57^KIP2−/− CFUe undergoing Dex-dependent self-renewal, relative to wild-type littermate culture. Representative of three independent experiments. (H) p57^KIP2 rescues p57^KIP2-deficient CFUe self-renewal. p57^KIP2-deficient S0 cells were transduced with low-titer virus (viral supernatant at the indicated dilutions) encoding p57^KIP2 or with empty vector. Cells transduced with p57^KIP2 showed significant improvement in self-renewal. Wild-type S0 cells transduced in parallel showed a reduction in self-renewal rate. (I) The CDK inhibitor drug roscovitine rescues self-renewal of p57^KIP2-deficient CFUe. p57^KIP2−/− S0 cells and wild-type S0 cells from littermate embryos were harvested and cultured in self-renewal medium containing Epo + Dex in the presence or absence of roscovitine. See also fig. S6B.