Flow cytometric assays of caspase-3 activity, cell-cycle changes, and cell proliferation assays in SALL4 knockdown NB4 cells. Two shRNA retroviral constructs that targeted different regions of the SALL4 were made, and their ability to reduce SALL4 mRNA in NB4 cells was confirmed by Q-RT-PCR (Figure S1). NB4 cells with approximately 75% reduction of SALL4 expression levels (via shRNA no. 7412) were used in these experiments. (A,D) NB4 cells transduced with the control retrovirus. (B,E) NB4 cells transduced with SALL4 shRNA retroviruses. (C,F) Bmi-1–expressing plasmid was transfected into SALL4 knockdown NB4 cells. Caspase-3 activity analysis shows shRNA-mediated reduction of SALL4 induces apoptosis in NB4 cells (A,B). SALL4 knockdown of NB4 cells can be rescued from apoptosis with ectopic expression of Bmi-1 (C). Cell-cycle changes and cellular DNA synthesis in control NB4 cells and SALL4 knockdown NB4 cells were monitored by BrdU-incorporation assay and analyzed by flow cytometry (3% background debris were excluded); data show knockdown of SALL4 induces cell-cycle arrest and decreased DNA synthesis (D,E). By ectopically expressing Bmi-1, SALL4 knockdown cells can be rescued from cell-cycle and DNA synthesis arrest (F). (G) Proliferation assay shows that SALL4 knockdown affects the growth rate of NB4 cells. Cells (5 × 10⁴) from stable clones expressing a control or SALL4 shRNA retrovirus (experiment) were plated in RPMI with 10% FBS. This assay was performed in duplicate and cell numbers were counted daily using a trypan blue–based assay.