Characterisation of differentiating Hoxb8 cells reconstituted with human wt or mutant NE.

<p>NE^-/- Hoxb8 neutrophils transduced with empty vector pMIGR1 (pMiGR1), human neutrophil elastase (hNE), or hNE mutant G185R (hNEG185R) (genetic background C57BL/6) were induced to undergo differentiation by estrogen withdrawal for 4 days. (A) Cell numbers of differentiating neutrophils were measured daily by automated counting with a CASY cell counter after initial seeding of 300,000 cells/well in a 6-well plate at day 0. Data are mean/SD of 3 independent experiments. (B) Cell death analysis of Hoxb8 neutrophils cultured from day 0 to day 4 of differentiation as described above. Viability was determined using a CASY cell counter. Data are mean/SD of 3 independent experiments. (C) Western blot analysis of human NE (hNE) and BIP (GRP78/HASP5) expression in cell lysates of Hoxb8 cells at day 2 of differentiation. Hoxb8 cells transduced with empty vector (pMIGR1), human wt NE (hNE) or human NE mutant G185R (hNEG185R) were induced to undergo differentiation <i>in vitro</i> by estrogen withdrawal. Differentiating cells were harvested on day 2. Samples were directly lysed in Laemmli buffer and heated at 95°C for 5 min. Samples were separated by SDS-PAGE, transferred onto nitrocellulose membranes and probed with antibodies against mNE and BIP followed by detection using enhanced chemoluminescence. GAPDH served as loading control.