SLPI is involved in a cell-cycle regulatory network. (A) Schematic diagram of the experimental design. BM CD34⁺ cells were transduced with SLPI-specific or control-RFP–tagged shRNA constructs and serum-starved for 24 hours. RFP⁺ cells were sorted, treated with G-CSF for 18 hours, and lysed in RNA lysis buffer. RNA from sorted cells was isolated and used for microarray analysis with Affymetrix GeneChip microarrays (Gene Expression Omnibus accession number GSE53353, National Center for Biotechnology Information tracking system number 16934073). (B-C) Microarray data were processed using GSEA software. Heatmaps show differences in mRNA expression of target genes involved in a cell regulatory network (B) and the c-Myc signaling pathway (C) in SLPI-deficient CD34⁺ cells compared with control-shRNA–transduced cells treated with G-CSF. Upregulation of mRNA expression is shown in red and downregulation in blue. (D) c-Myc, survivin, and cyclin D1 mRNA expression in CD34⁺ cells transduced with shRNA constructs as indicated in panel A and treated with G-CSF. c-Myc, survivin, and cyclin D1 mRNA expression, normalized to that of β-actin and presented in arbitrary units (AUs), was measured by qRT-PCR; data represent mean ± SD of triplicates. (E) Expression of c-Myc, CDC25, cyclin B, cyclin D1, cyclin E2, and CDK6, CDK3, CDK4 mRNA in BM CD33⁺ myeloid cells of studied groups. CN, severe congenital neutropenia; ctrl, control; IN, idiopathic neutropenia. mRNA expression of indicated above target genes, normalized to that of GAPDH and presented in AUs, was measured by qRT-PCR; data represent mean ± SD of triplicates (*P < .05; **P < .01).