Detection of apoptosis in U937 cells. (A) Dual staining of FITC-labeled Annexin-V and PI. To detect apoptotic cells, U937 cells were transfected with 500 ng of control siRNA and Ubc9 siRNA by AMAXA nucleofection and treated with RA. After the 48-hour time point, cells were stained with Annexin-V and PI. Numbers in the lower right quadrant of each plot represent the percentage of cells in early apoptosis (Annexin-V⁺ and PI⁻). Numbers in the top right quadrant of each plot represent the percentage of dead cells (Annexin-V⁺ and PI⁺). (B) The histogram represented the values calculated for the Annexin-V staining for the above experimental settings. Values shown here from 3 independent experiments represented as means (± SEM). (C) Western blot analysis performed for the expression of Ubc9 in U937 cells at different time points after overexpression of Ubc9 siRNA and a nonsilencing siRNA control.