SLPI is essential for granulocytic differentiation in vitro. CD34⁺ BM cells were transduced with lentiviral constructs of 2 different SLPI-specific RFP-tagged shRNAs (SLPI shRNA 1 and SLPI shRNA 2) or with irrelevant shRNA (ctrl shRNA). On day 4 of culture, RFP⁺ cells were sorted and in vitro granulocytic differentiation was tested. (A) β-Actin-normalized SLPI mRNA expression, in arbitrary units (AUs), in sorted RFP⁺ NB4 cells transduced with shRNA constructs as indicated above was measured by qRT-PCR; data represent mean ± SD of triplicates (*P < .05 vs control lv samples). (B) Representative western blot images showing SLPI protein expression in total lysates from RFP⁺ cells transduced with shRNA constructs as indicated above. Ponceau staining is depicted as a loading control. (C-D) Granulocytic differentiation of transduced CD34⁺ cells was tested using a liquid culture method. The percentage of CD11b⁺, CD15⁺, and CD16⁺ cells in the RFP⁺ cell population on day 14 of culture is presented. Data represent mean ± SD of triplicates of the median of 2 different SLPI shRNAs (SLPI shRNA1 and SLPI shRNA2) (*P < .05). (E-F) CD34⁺ BM cells, transduced with SLPI-specific or control-GFP–tagged shRNA constructs, were labeled with BrdU for 30 minutes, after which the percentage of BrdU⁺ cells (E) and cell-cycle profile (F) were assessed using a BrdU Flow Kit and FACS analysis, respectively. Data represent the median of 2 different samples of cells transduced with either SLPI-specific shRNA 1 or shRNA 2 (mean ± SD of triplicates of 3 experiments; *P < .05). (G) The apoptosis of CD34⁺ BM cells, transduced with SLPI-specific or control-GFP–tagged shRNA, was quantified by flow cytometry using an annexin V apoptosis detection kit. Data are expressed as the percentage of annexin V⁺ cells in the GFP⁺ cell population. Data represent the median of 2 different samples of cells transduced with either SLPI-specific shRNA 1 or shRNA 2 (mean ± SD of triplicates of 3 experiments; *P < .05). (H) CD34⁺ cells from healthy individuals (n = 3) were transduced with SLPI-specific or control-GFP–tagged shRNA lentiviral constructs, and GFP⁺ cells were sorted on day 4 of culture. The indicated CFU assays were performed. Data are mean ± SD and are derived from 2 independent experiments, each in duplicate (**P < .01).