Knockdown of Ubc9 expression in CD34 + and U937 leads to enhanced granulocytic differentiation. (A,B) Flow cytometric analysis performed for CD15 expression at day 6 (CD34⁺ cells) and day 3 (U937 cells) after the cotransfection of Ubc9 siRNA, a control siRNA and C/EBPαp30 in CD34⁺ and U937 cells and induced with G-CSF and RA, respectively. The fold changes are expressed as percentages and shown as a dotplot representative of 1 experiment. siRNA control was also used in all the experiments and is shown. (C,D) The percentage of the population of CD15⁺ cells from 3 independent experiments are represented as means (± SEM), with P values shown as histograms. (E) The percentage of the population of CD15⁺ cells after HA-Ubc9 overexpression in U937 cells and RA treatments. (F-I) Quantitative real-time PCR for the expression of G-CSF receptor and MPO. The fold increase for expression was calculated using Δct = (Ct sample − Ct control), and ΔCt values for each sample were standardized by GAPDH Ct value. The fold change was calculated as (= 2^−Δct). Values are expressed as means (± SEM) for 3 independent experiments, with P values shown on histograms. The transfection efficiency in U937 cells and CD34⁺ cells after GPF nucleofection technology was calculated to be 80% and 50%, respectively (Figure S2B,C).