PIM2 helps to stabilize cytoplasmic p21^Cip1 and promotes the caspase 3/p21^Cip1 interaction. (A) Left: CDKN1A mRNA expression was measured on D6 in CD23⁺ aBCs and PBs. Data are presented as the median (range), n = 10. Right: PIM2 and p21^Cip1 protein expression on D6 in CD23⁺ aBCs and PBs. (B) PIM2 and p21^Cip1 protein expression was measured in nuclear and cytoplasmic fractions from CD23⁺ aBCs and PBs. (C) PIM2 (left) and p21^Cip1 (right) immunoprecipitation (IP) in PBs. Heat shock protein 90β (HSP90β), p21^Cip, and PIM2 were detected by immunoblotting. (D) PIM2 and p21^Cip1 protein expression after treatment with SSO-PIM2 (top) or PIMi (bottom) in total extracts from PBs. (E) PBs were sorted and then treated with PIMi and incubated with cycloheximide (CHX). At indicated times, p21^Cip1 expression was assessed by immunoblotting. (F) PBs were sorted and then treated with PIMi for 2 hours in the presence (or not) of MG-132. p21^Cip1 was assessed by immunoblotting. (G) PBs were treated for 30 minutes with PIMi after incubation in the presence of MG-132 for 2 hours. Polyubiquitinated p21^Cip1 and native p21^Cip1 were assessed by immunoblotting. (H) FRET/FLIM (Förster resonance energy transfer (FRET) by fluorescence lifetime imaging (FLIM)) was analyzed in XG21 cells stained for caspase 3 (the donor) in the presence or absence of p21^Cip1 (the acceptor). Cells were left untreated (upper panels) or treated with PIMi (lower panels). The graph shows the quantified ΔLifetime. Data are presented as the median (range), n = 20 cells per condition. Pseudocolor scale, pixel-by-pixel ΔLifetime. Scale bar: 10 µm. (I) p21^Cip1 (top) and caspase 3 (bottom) IP in PBs. Caspase 3 and p21^Cip were detected by immunoblotting. (J) p21^Cip1, procaspase 3, cleaved caspase 3, and cleaved PARP protein expression on D6 in PBs, after transfection with siCDKN1A or siCTL on D4. (K) On D4, activated B cells were treated for 24 hours with increased doses of p21i in the presence or absence of Q-DEVD-OPH. PB viability (the proportion of active caspase 3–negative cells) was assessed by flow cytometry. Data are presented as the mean ± SD, n = 5. (L) The PIM2/p21^Cip1/caspase 3 pathway in PBs. p21^Cip1 is stabilized in the cytoplasm by a protein complex that also includes PIM2 and HSP90β. Due to this localization, p21^Cip1 interacts with procaspase 3 and blocks its activation. Statistical significance was evaluated by using Mann-Whitney U (panels A and H) and Kruskal-Wallis (panel K) tests. *P < .05, ***P < .001, ****P < .0001. Cl, cleaved; DMSO, dimethyl sulfoxide; IgG, immunoglobulin G; IP-Ub, immunoprecipitation of ubiquitin forms; ns, not significant; NT, no treatment; siCTL, small interference (si) RNA control (CTL). Further details are presented in supplemental Figure 5.