Fig. 1Effect of PP2 on all-trans-retinoic acid (ATRA)- or arsenic trioxide (ATO)-induced differentiation of NB4 cells. NB4 cells were treated with 10 µM of PP2 alone, 0.5 µM of ATO alone, 0.001 µM of ATRA alone, 0.5 µM of ATO plus 10 µM of PP2, or 0.001 µM of ATRA plus 10 µM of PP2 for 72 hours. Cells were harvested, incubated with anti-CD11b-PE antibody or isotype-matched antibody, and analyzed by flow cytometry. A total of 10,000 cells per sample were analyzed for expression of CD11b. (A) Open histograms, cells stained with isotype control antibody; shaded histograms, staining with CD11b antibody. Values in the upper right corner illustrate the percentage of CD11b-positive cells. (B) Columns represent the averages for five independent experiments with similar results. Control cells that were not treated with any reagents. a)p<0.05 significantly higher than ATO alone, b)p<0.05 significantly higher than ATRA alone, c)p<0.05 significantly higher than PP2 combined with ATO.
Fig. 2Effect of PP2 on all-trans-retinoic acid (ATRA)- or arsenic trioxide (ATO)-induced granulocytic differentiation of NB4 cells. NB4 cells were treated with 10 µM of PP2 alone, 0.5 µM of ATO alone, 0.001 µM of ATRA alone, 0.5 µM of ATO plus 10 µM of PP2, or 0.001 µM of ATRA plus 10 µM of PP2 for 72 hours. After treatment for 72 hours, cells were evaluated for granulocytic differentiation on the basis of nitroblue tetrazolium reduction assay. Using a light microscope, a minimum of 200 cells were counted, in order to determine the percentage of differentiated cells. (A) Differentiated cells were identified by their intracellular blue formazan deposits. (B) Columns represent the averages for four independent experiments with similar results. C indicates control that cells were not treated with any reagents. a)p<0.05 significantly higher than ATO alone, b)p<0.05 significantly higher than ATRA alone, c)p<0.05 significantly higher than PP2 combined with ATO.
Fig. 3Effect of PP2 on all-trans-retinoic acid (ATRA)- or arsenic trioxide (ATO)-induced apoptosis of NB4 cells. NB4 cells were treated with 10 µM of PP2 alone, 0.5 µM of ATO alone, 0.001 µM of ATRA alone, 0.5 µM of ATO plus 10 µM of PP2, or 0.001 µM of ATRA plus 10 µM of PP2 for 72 hours. After treatment for 72 hours, cells were stained with Annexin V-FITC and analyzed by flow cytometry (results representative of three independent experiments). Values in the center present the percentage of apoptotic cells.
Fig. 4Retinoic acid-induced gene expression. NB4 cells were treated for 72 hours with 10 µM of PP2 alone, 0.5 µM of arsenic trioxide (ATO) alone, 0.001 µM of all-transretinoic acid (ATRA) alone, 0.5 µM of ATO plus 10 µM of PP2, or 0.001 µM of ATRA plus 10 µM of PP2. Fifty micrograms of whole cell lysates were resolved by sodium dedecyl sulfate polyacrylamide gel electrophoresis and subjected to western blotting with antibodies against intercellular adhesion molecule-1 (ICAM-1) or cathepsin D. Equal loading was determined by actin. Similar results were obtained in three independent experiments.