Figure 5.

Derepression of the Il7r gene increases IL7R expression and promotes downstream signaling. (A) IL-7 receptor expression of FACS-sorted pro-B cells (c-KIT⁺CD25^-) of miR-15a/b^{fl/fl Vav-Cre} and control mice as analyzed by quantitative PCR. (B, C) Flow cytometric analysis of IL-7 receptor surface expression in pro- (c-KIT⁺CD25^-), intermediate- (c-KIT^-CD25^-) and pre-B cells (c-KIT^-CD25⁺) from control or miR-15a/b^{fl/fl Vav-Cre} mice (B) as well as in vitro cultured progenitors thereof (C). For the in vivo data, the MFI values were normalized to the mean IL-7 receptor expression of control mice for each respective experimental day. (D) Western blot analysis of control and miR-15a/b^{fl/fl Vav-Cre} progenitors depicting pAKT^Ser473, AKT and GAPDH protein levels. (E) Representative histograms of flow cytometric analysis of intracellular pAKT^Ser473 levels in control and miR-15a/b^{fl/fl Vav-Cre} progenitors. The bar graph summarizes the pAKT^Ser473 MFI normalized to the respective Isotype. (F) Representative FACS plots depicting the “proliferating” progenitors defined as the FSC^hi population (dark grey) upon culturing the cells with 5 ng/ml, 1 ng/ml or 0.1 ng/ml IL-7 for 48 hours. Bar graphs summarize the quantification of the FSC^hi population among all living progenitors derived from miR-15a/b^{fl/fl Vav-Cre} or control mice. (G) Summary of the percentage of FSC^hi cells among pro-, intermediate and pre-B cells upon culturing the progenitors with 0.1 ng/ml IL-7 for 48 hours. MiR-15a/b^{fl/fl Vav-Cre} progenitors were statistically compared to miR-15a/b^{+/+ Vav-Cre} progenitors by an unpaired two-tailed Student’s t-test. *P < 0.05; **P < 0.005; ***P < 0.0005.