Figure 6. The functionally coupled H_v1 and Nox2 are induced in parallel, but largely independently during granulocytic differentiation in PLB-985 cells.

(a) Normal H_v1 expression can be induced in the absence of normal Nox2 level. For WB total cell lysates of 10⁶ cells were loaded each lane. Samples of PLB-985 cells were prepared before (0) and 2, 4, 6 days after inducing differentiation with 0.5% DMFA. In a separate sample (7*) cells were differentiated for 7 days, and DMFA treatment was applied in low-serum culture medium (0.5% v/v) to increase the differentiation pressure. Different anti-Nox2 labeled bands correspond to different glycozilation states of the 65 kDa Nox2 protein. (b) The normal expression of different phox subunits (Nox2, p22^phox and p47^phox) is not disturbed by strongly reduced H_v1 expression in differentiated PLB-985 cells. (c) Amongst differentiated PLB-985 clones amphotericin B amplifies superoxide production (2.92±0.48 times at 15 min, p<0.05, Mann-Whitney U test) only in clone F2, in which H_v1 expression is strongly diminished. No significant change in superoxide production was detected in E9, G9 and B7 clones in the presence of amphotericin B. Diogenes^™ reagent was used to detect the extracellular release of superoxide. Cells were preincubated for 15 min in a 1∶1 mixture of Diogenes^™ and H-medium with or without 10 µg/ml amphotericin B. At time point 0 cells were activated by 200 nM PMA. Negligible Diogenes^™ luminescence could be detected in PMA-treated, non-differentiated PLB-985 clones and during the preincubation period (not shown).