Extrinsic stimulations result in proliferation, NF-κB activation and in an unbalanced modulation of the Bcl-2 family in primary MCL cells. (A) qRT-PCR analysis of MKI67 from: left, primary MCL cells concomitantly isolated from LO (Pts 5 and 22: lymph node, Pt16: spleen) and PB; right, PB primary MCL cells cocultured on an L-40L layer in the presence of cytokines (D7) and relative to PB. (B) Immunoblotting of cell-cycle related proteins (pRb, Rb, PCNA) in primary CD5⁺ cells isolated from healthy donor (CBBC) or MCL patients (PB) at D0 and after coculture on L-40L+Ck at the time indicated (D3, D7). (C) qRT-PCR analysis of NF-κB target genes (IL2RG, PLEK, and CD74)³⁵  in primary MCL cells cultured as in panel A. Maver-1 and JeKo-1 were used as NF-κB signaling positive and negative controls, respectively.⁴  (D) Immunoblotting of classical (p-IκBα) and alternative (p52) NF-κB pathway proteins in primary MCL cells as in panel B. (E) qRT-PCR analysis of indicated genes in primary MCL cells cultured as in panel A. (F) Immunoblotting of Bcl-2 family proteins in primary MCL cells as in panel B. *Highlights Bim underexposure resulting from high expression in sample 18. (G) Immunoblotting analysis of indicated proteins in primary MCL cells concomitantly isolated from LO and PB. Quantification of protein level was normalized to actin and indicated below each protein.