Spi-B is required for CAL-1 cell survival and proliferation. (A) CAL-1 cells were transduced with Spi-B shRNA or control renilla shRNA and cultured for 14 days. Cell growth was determined by flow cytometric analysis to measure the percentages of GFP⁺ cells every 3 days. (B) Mean values of 3 experiments in which GFP⁺ cell percentages were determined at 13 days after transduction. Values were normalized to the percentage of GFP⁺ cells at day 2 after transduction (A; *P = .026). (C) CAL-1 cells transduced with Spi-B shRNA or control renilla shRNA were stained with the CellTrace violet proliferation kit. Mean fluorescence intensity (MFI) was followed for 3 days by flow cytometry. (D) To assay for apoptosis, annexin V–PE and 7-AAD double staining was performed on CAL-1 cell transduced with Spi-B shRNA or control renilla shRNA 5 days after sorting GFP⁺ cells. Numbers represent percentages of early apopotic cells (annexin V⁺7-AAD⁻) and late apoptotic/necrotic cells (annexin V⁺7-AAD⁺) in the indicated gates. One representative experiment of 3 is displayed. (E) The percentages of apoptotic cells were measured over time in Spi-B shRNA or control renilla shRNA transduced cells after sorting GFP⁺ cells. One representative experiment is shown of 2. (F) RAG-2^−/−γc^−/− immunodeficient mice were injected subcutaneously in the right and left flank with 0.25.10⁶ Cal-1 cells expressing Spi-B shRNAs or renilla shRNAs. Tumor growth is shown as absolute cell numbers measured 20 days after engraftment (*P = .037). Each dot/square represents 1 mouse.