Enforced MCL1 expression blocks BCR-induced programmed cell death in B-NHL cells. (A) Analysis of BCR-induced programmed cell death in MEC1 B cells. MEC1 B cells were transduced with lentiviruses containing MCL1 shRNAi. After infection, GFP⁺ B cells were purified using Flow Sorter (FACSAria BD), whereas untransduced MEC1 B cells were used as negative controls. Programmed cell death on BCR stimulation was performed and evaluated as described in Figures 1B and 2B. (B) Analysis of BCR-induced programmed cell death in MCL1 overexpressing B-cell lymphomas. RAMOS RA-1 B cells were transduced with retroviruses (using the pLZR-IRES-GFP backbone⁶⁸ ) containing empty vector or Flag-murine Mcl1 cassette. The Flag-Mcl1 construct was cloned from pCMV-3xFlag-Mcl1 (3xFlag epitope tag cloned in frame with mouse Mcl-1 cDNA including ATG) into the EcoR1 site of the pLZR-IRES-GFP retroviral vector. Retroviruses were pseudo-typed with amphotropic envelopes using the envelope-expressing packaging cell line BING-CAK8 (ATCC). After infection, GFP⁺ B cells were purified (∼80% of enrichment) using Flow Sorter (FACSAria BD), whereas untransduced RAMOS RA-1 B cells were used as negative controls. Programmed cell death on BCR stimulation was performed and evaluated as described in Figures 1B and 2B. (C) Western blot for murine Mcl1 in total protein extracts (RIPA extraction) from GFP⁺ purified MCL1 overexpressing and empty vector (negative control) transduced RAMOS RA-1 B cells. Untransduced RAMOS RA-1 B cells were used as negative control as well. *Background band that can be used as loading control.