Inhibition of NE leads to diminished levels of SLPI. (A-D) The leukemia cell lines NB4 and HL-60 and BM CD34⁺ cells were transduced with lentiviral constructs of 2 different ELANE-specific RFP-tagged shRNAs (ELANE shRNA E3 and ELANE shRNA E7) or with irrelevant shRNA (ctrl shRNA). On day 4 of culture, RFP⁺ cells were sorted and analyzed. (A) Representative western blot images showing NE protein expression in total lysates from NB4 cells transduced with shRNA constructs, as indicated above. α-Tubulin staining was used as a loading control. (B) β-Actin–normalized SLPI mRNA expression, in arbitrary units (AUs), in NB4 and HL-60 cell lines transduced with 2 different ELANE-specific shRNAs (ELANE shRNA E3 and ELANE shRNA E7) or with irrelevant shRNA (ctrl shRNA) was measured by qRT-PCR; data represent mean ± SD of triplicates (*P < .05 vs control lv samples). (C) Representative western blot images of SLPI in NB4 cells transduced with shRNA constructs, as indicated above. β-Actin was used as a loading control. (D) β-Actin–normalized SLPI mRNA expression, in AUs, in BM CD34⁺ cells transduced with 2 different ELANE-specific shRNAs (ELANE shRNA E3 and ELANE shRNA E7) or with irrelevant shRNA (ctrl shRNA) was measured by qRT-PCR; data represent mean ± SD of triplicates (*P < .05 vs control lv samples). (E) SLPI mRNA expression in NB4 cells, transduced with HAX- or HCLS1-specific shRNAs or with irrelevant shRNA (ctrl shRNA). mRNA expression, normalized to that of β-actin and presented in AUs, was measured by qRT-PCR; data represent mean ± SD of triplicates (*P < .05). (F) β-Actin–normalized SLPI mRNA expression, in AUs, in the NB4 cell line, transduced with LEF-1–specific shRNA or irrelevant shRNA (ctrl shRNA) was measured by qRT-PCR; data represent mean ± SD of triplicates (*P < .05 vs control lv samples). (G) Three LEF-1 binding sites on the promoter (blue ovals) were mapped to the region of the SLPI promoter using in silico analysis. Quantitative ChIP assay of nuclear extracts of NB4 cells and PCR products were amplified using qRT-PCR with primer pairs flanking the C/EBPα binding sites (b. s. 1; b. s. 2; b. s. 3) of the SLPI promoter to assess C/EBPα binding to the SLPI promoter. Ab, antibody; C/EBPα, ChIP with anti-C/EBPα antibody; isotype, isotype antibody control. Bar graphs represent % of ChIP to input chromatin of target regions. Data are mean ± SD derived from 2 independent experiments.