Enforced expression of

CUL-4A inhibits granulocytic differentiation. (A) The cell lines generated from PLB-985 cells transfected with pEF-PGKpac (empty vector) or with pEFpac-CUL-4A-HA (CUL-4A-HA1 or CUL-4A-HA2) were induced with DMF to differentiate into granulocytes for 0 to 6 days, and total RNA was isolated from a sample taken at each time point. RNA (5 μg) from each sample was electrophoresed, and the amount of gp91^phox mRNA was determined by Northern blot analysis and normalized with respect to the amount of actin mRNA. The average of results from 2 independent experiments appears in the graph to the right of the autoradiogram (empty vector control, ▪;CUL-4A-HA1, ●; CUL-4A-HA2, ▴). 100% corresponds to the amount of gp91^phox mRNA measured in control cells 6 days after induction. Error bars denote SEM. For data points with no visible error bars, the error bars are smaller than the symbol. (B) The 3 cell lines were induced with DMF as described for panel A, and total protein was isolated from a sample taken at each time point. Protein (10 μg) from each sample was electrophoresed, and the amount of gp91^phoxprotein was determined by immunoblot and normalized with respect to the amount of actin. The results from a representative experiment are shown. The symbols are as described for panel A. 100% corresponds to the amount of gp91^phox protein measured in control cells 6 days after induction. (C) The 3 cell lines were induced with DMF for 4 days, and cells with oxidase activity were detected by their ability to reduce nitro blue tetrazolium to generate violet formazan granules. Cells with oxidase activity were counted and the results from 3 independent experiments were graphed. Error bars represent SEM, which is reported in the text. Comparing eitherCUL-4A-HA1 or CUL-4A-HA2 and the control,P < .01 by Student t test. (D) The 3 cell lines were induced with DMF as described above and slide preparations were stained with Diff-Quick Stain. Cells with band or polymorphonuclear nuclear morphology were counted, and the results of 3 independent experiments were graphed. Error bars represent SEM, which is reported in the text. Comparing either CUL-4A-HA1 or CUL-4A-HA2 and the control, P = .0002 by Student t test. (E) Fields representative of 3 separate experiments graphed in panel D were photographed (magnification × 40 and × 100 for inset images) and appear as follows: (i) empty vector control cells before DMF addition, (ii) empty vector control cells 4 days after induction, (iii) CUL-4A-HA1 cells 4 days after induction, and (iv) CUL-4A-HA2 cells 4 days after induction. Arrows in panel i point to examples of cells with undifferentiated cell morphology, while those in panel ii show differentiated cells.