Figure 6.
PIM2 in BM plasma cells. (A) PIM2 mRNA expression in different tonsil-derived B-cell populations (n = 7) and in BM PCs (n = 9). The data are presented as the median (range). (B) Immunofluorescence staining of PIM2 (green) and CD138 (orange) on paraffin-embedded normal BM. 4′,6-Diamidino-2-phenylindole (blue) stains the nucleus. Scale bar: 20 µm. (C) PIM2 mRNA and PIM2 protein expression in CD23+ aBCs and PBs on D6 and ePCs on day 10 (D10). Data are presented as the median (range), n = 10. (D) On D10, ePCs were sorted, starved, and treated (or not) with an anti–IL6R monoclonal antibody 1 hour before stimulation. IL-6 and supernatants of BM mesenchymal stroma cells (s-MSC) were pre-incubated (or not) for 1 hour with an anti-IL6 monoclonal antibody. PIM2 mRNA expression (left) and protein expression (right) in ePCs 5 hours after the addition of IL-6 or s-MSC. Data are presented as the mean ± SD, n = 4. (E) Immunofluorescence staining of PIM2 (green), CD41 (red), and CD138 (orange) on paraffin-embedded normal BM. 4′,6-Diamidino-2-phenylindole (blue) stains the nucleus. Scale bar: 20 µm. (F) On day 9, cells were treated with PIMi. The proportion of active caspase-3–positive cells was assessed on D10 by using flow cytometry. Data are presented as the mean ± SD, n = 6. (G) On D10, live cells were treated for 2 hours with PIMi. Protein expression levels of caspase 9, XIAP, caspase 3, and p21Cip were assessed by immunoblotting. (H) p21Cip1 expression was assessed on D10 in nuclear and cytoplasmic fractions by immunoblotting and immunofluorescence. Sytox (blue) stains the nucleus. Scale bar: 5 µm. (I) FRET/FLIM data were analyzed for ePCs stained for (the donor) PIM2 (left) or caspase 3 (right) in the presence or absence of p21Cip1 (the acceptor). 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. Statistical significance was evaluated by using the Mann-Whitney U test in panels A, C, D, F, and I. *P < .05, **P < .01, ***P < .001, ****P < .0001. DMSO, dimethyl sulfoxide; FRET/FLIM, förster resonance energy transfer (FRET) by fluorescence lifetime imaging (FLIM)FSC, forward scatter; GC, germinal center; MBC, memory B cells; NBC, naive B cells; ns, not significant; NT, no treatment. Further details are presented in supplemental Figure 6.

PIM2 in BM plasma cells. (A) PIM2 mRNA expression in different tonsil-derived B-cell populations (n = 7) and in BM PCs (n = 9). The data are presented as the median (range). (B) Immunofluorescence staining of PIM2 (green) and CD138 (orange) on paraffin-embedded normal BM. 4′,6-Diamidino-2-phenylindole (blue) stains the nucleus. Scale bar: 20 µm. (C) PIM2 mRNA and PIM2 protein expression in CD23+ aBCs and PBs on D6 and ePCs on day 10 (D10). Data are presented as the median (range), n = 10. (D) On D10, ePCs were sorted, starved, and treated (or not) with an anti–IL6R monoclonal antibody 1 hour before stimulation. IL-6 and supernatants of BM mesenchymal stroma cells (s-MSC) were pre-incubated (or not) for 1 hour with an anti-IL6 monoclonal antibody. PIM2 mRNA expression (left) and protein expression (right) in ePCs 5 hours after the addition of IL-6 or s-MSC. Data are presented as the mean ± SD, n = 4. (E) Immunofluorescence staining of PIM2 (green), CD41 (red), and CD138 (orange) on paraffin-embedded normal BM. 4′,6-Diamidino-2-phenylindole (blue) stains the nucleus. Scale bar: 20 µm. (F) On day 9, cells were treated with PIMi. The proportion of active caspase-3–positive cells was assessed on D10 by using flow cytometry. Data are presented as the mean ± SD, n = 6. (G) On D10, live cells were treated for 2 hours with PIMi. Protein expression levels of caspase 9, XIAP, caspase 3, and p21Cip were assessed by immunoblotting. (H) p21Cip1 expression was assessed on D10 in nuclear and cytoplasmic fractions by immunoblotting and immunofluorescence. Sytox (blue) stains the nucleus. Scale bar: 5 µm. (I) FRET/FLIM data were analyzed for ePCs stained for (the donor) PIM2 (left) or caspase 3 (right) in the presence or absence of p21Cip1 (the acceptor). 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. Statistical significance was evaluated by using the Mann-Whitney U test in panels A, C, D, F, and I. *P < .05, **P < .01, ***P < .001, ****P < .0001. DMSO, dimethyl sulfoxide; FRET/FLIM, förster resonance energy transfer (FRET) by fluorescence lifetime imaging (FLIM)FSC, forward scatter; GC, germinal center; MBC, memory B cells; NBC, naive B cells; ns, not significant; NT, no treatment. Further details are presented in supplemental Figure 6.

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