PIM2 supports cell cycle entry by promoting the G1/S transition. (A) Flow cytometry evaluation of the cell cycle in PBs on D6 after treatment with SSO-PIM2 and increasing doses of PIMi, compared with controls (n = 6). (B) Left: CDC25A mRNA expression in CD23⁺ aBCs and PBs on D6. Data are presented as the median (range), n = 10. Right: CDC25A protein expression (in the red square) in CD23⁺ aBCs and PBs on D6 after treatment with PIMi. †Nonspecific band. (C) D4 B cells were treated with PIMi and incubated with cycloheximide (CHX). At the indicated time points, CDC25A expression was analyzed by immunoblotting. (D) On D4, B cells were treated with PIMi and incubated in the presence of MG-132 for 2 hours. CDC25A expression was analyzed by immunoblotting. (E) Left: CDKN1B mRNA expression in CD23⁺ aBCs and PBs on D6. Data are presented as the median (range), n = 10. Right: PIM2, p(Thr198)-p27^Kip1 (p-p27), and total p27^Kip1 protein expression in CD23⁺ aBCs and PBs on D6 in total protein extracts and nuclear and cytoplasmic fractions. (F) Cells were incubated with CHX on D6, just before sorting. At the indicated time points, p27^Kip1 expression in CD23⁺ aBCs and PBs was analyzed by immunoblotting. (G) Cells were incubated for 4 hours with MG-132 before sorting on D6. p27^Kip1 expression in CD23⁺ aBCs and PBs was assessed by immunoblotting. (H) Left: Immunoprecipitation (IP) of p27^Kip1 in D4-activated B cells. PIM2 and p27^Kip1 were detected by immunoblotting. Right: On D4, activated B cells were shortly treated with PIMi. At the indicated time points, p-p27 and total p27^Kip1 protein expression were assessed by immunoblotting. (I) PIM2 and p27^Kip1 protein expression in PBs after treatment with PIMi in total protein extracts and nuclear and cytoplasmic fractions. (J) On D6, cells were treated with PIMi and incubated with CHX before sorting. At the indicated time points, p27^Kip1 expression in PBs was assessed by immunoblotting. (K) On D6, cells were treated with PIMi for 2 hours before sorting. p-p27 and total p27^Kip1 were assessed by immunoblotting. (L) On D6, cells were incubated in the presence or absence of PIMi for 2 hours before the addition (or not) of MG-132 for 4 hours. Left: p27^Kip1 was assessed by immunoblotting. Right: Polyubiquitinated p27^Kip1 and native p27^Kip1 were assessed by immunoblotting. (M) PIM2 promotes the G1/S transition. Positive effects (in pink) are PIM2 dependent, whereas effects not directly related to PIM2 are shown in green. On one hand, PIM2 induces p27^Kip1 degradation once the protein is localized in the cytoplasm; on the other hand, PIM2 maintains the expression of CDC25A. Statistical significance was evaluated by using Kruskal-Wallis tests (panel A, for PIMi experiment) and Mann-Whitney U test (panels A [for SSO experiment], B, and E). *P < .05, **P < .01, ****P < .0001. DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; IP-Ub, immunoprecipitation of the ubiquitin forms; NT, no treatment; ns, not significant. Further details are presented in supplemental Figure 3.