Re-expression of candidate genes into 6q-deleted NK cell lines. (A) Schema outlining gene transduction and induction using the Tet-Off system. The Retro-X Tet-Off Advanced inducible expression system (Clontech) was used. The Tet-controlled transactivator (tTA) was initially transduced by retroviral infection, and was followed by neomycin (G418) selection (Neo). Selected cells were cloned by limiting dilution, and the clone with the highest induction efficiency was isolated. cDNA of the gene of interest (GOI) was then inserted into pRetroX-Tight-Pur and transduced into the isolated clone. After puromycin selection (Pur), doxycycline was removed and cells were observed for 6 days. The Western blot shown is representative of the result obtained after incubation of anti-FLAG antibody with the cellular extract from the FLAG-PRDM1–transduced cell line. TRE indicates tetracycline response element. (B) Induction efficiency of the Tet-Off system was analyzed using pRetroX-Tight-Pur-GFP. pRetroX-Tight-Pur-GFP was transduced and selected as described in panel A. More than 95% of the cells showed induced GFP expression. (C) Western blot analysis. Gene expression was induced as described in panel A. A total of 1 × 10⁶ cells was resuspended in 100 μL of 2× sample buffer (125mM Tris, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.02% bromophenol blue) and 10 μL was subjected to PAGE. After transfer of the separated proteins onto the membrane, the membrane was incubated with anti-FLAG antibody for FOXO3, PRDM1, ATG5, HACE1, LACE1, and PREP, and with anti-A20 antibody for A20 (top panel). Anti-actin antibody was then incubated with the membrane after stripping to measure cellular extract quantity (bottom panel). The molecular size marker 97 kDa is indicated by an arrowhead. (D) Time-course changes with FOXO3 and PRDM1 re-expression. Cell samples at days 0, 1, 2, and 5 were analyzed as described in panel C. Each band was converted to a numerical value using ImageJ software, and the ratio to actin was calculated and is shown below each band. (E) Effect of re-expressed PRDM1 or FOXO3 on NKL. A total of 1 × 10⁵ cells was resuspended in medium with or without doxycycline (day 0). On day 2, cells were diluted by up to 10-fold and new medium was added because the cells had reached confluence. The horizontal axis indicates the time elapsed after doxycycline removal. The vertical axis indicates the average and standard deviation of the cell number ratio (Dox−/Dox+). Cell numbers were determined using the trypan blue dye-exclusion assay. Experiments were performed in triplicate. Cell proliferation was strictly suppressed on day 6 when FOXO3 and PRDM1 were re-expressed, unlike A20. (F) Each candidate gene, PRDM1 mutant (PRDM1^{174STOP and} PRDM1^203STOP), and FOXO3 mutant (FOXO3^P8R and FOXO3^I646F) was induced as described in panel D. Medium change and dilution were performed on day 2 and cell counts were determined on day 6. The vertical axis indicates the ratio of cell number with and without doxycycline. Experiments were performed in triplicate and standard deviations and average scores are described. Candidate genes, except in the case of FOXO3 and PRDM1, had no effect on cell proliferation, although these genes were induced and protein expression was confirmed. *Significantly lower than vector control (P < .05). (G) Effect of re-expressed PRDM1 or FOXO3 on SNK10. A total of 1 × 10⁵ cells was resuspended in medium with or without doxycycline (day 0) and analyzed as described in panel D. On day 2, cells were diluted 5-fold. Cell proliferation was suppressed on day 6 when FOXO3 and PRDM1 were re-expressed, whereas the control genes (vector control and GFP) had no effect. *Significantly lower than vector control (P < .05).