shRNAi lentiviral screen in RAMOS RA-1 B-NHL cells. (A) Lentiviral (HIV)-based screen for BCR regulatory molecules. RAMOS RA-1 B cells were transduced with individual lentiviruses from the shRNAi lentiviral mini-library (Table S1) pseudo-typed with vesicular stomatitis glycoprotein (VSVG) as described in “Lentiviral-mediated shRNAi transfer.” After lentiviral infection, transduced B cells were assayed for BCR-induced programmed cell death as described in Figure 1B. Survival index (SI) for each individual transduction was calculated as specified in Figure 2B. Representative SI value from 150 shRNAi lentiviral clones is shown. (B) Effect of scramble RNAi in the threshold of BCR-induced programmed cell death. RAMOS RA-1 B cells were transduced with lentiviruses containing a scramble shRNAi (negative control). SI value was calculated according to the formula described in the figure. Data for scramble shRNAi lentivirus infections are representative of 5 independent experiments. Scramble sequence included GAGACACCAAAGCATACAA. (C) Western blot for human MCL1 and Actin (loading control) in total protein extracts (RIPA extraction) from GFP⁺ purified MCL1 shRNAi, scramble shRNAi and empty vector-transduced RAMOS RA-1 B cells. GFP⁺ B cells were purified using Flow Sorter (FACSAria BD).