Figure 6

siRNA transfection of U937 cells. A. U937 cells (control U937) and cells transfected with a FITC-conjugated Negative Control siRNA AF488 (FITC-siRNA-transfected U937) were analyzed by FACS and revealed more than 95% transfection efficiency by this method. B. Western blot analysis of U937 control cells (U937), U937 cells transfected with a negative control siRNA (neg. siRNA) and U937 cells transfected with a VCP/p97 siRNA (VCP siRNA) were analyzed for VCP/p97 protein expression 72 h after the transfection and in the presence of 10 nM TPA for 24 h (+TPA), respectively. C, D. cDNA microarray analysis of VCP/p97 siRNA-transfected U937 cells during TPA-induced differentiation. U937 cells transfected with either negative control siRNA or VCP/p97 siRNA in the presence or absence of 10 nM TPA for 24 h, respectively, were subjected to cDNA microarray analysis provided by the Stanford Functional Genomics Facility. U937 control transfectants were compared to VCP/p97 transfectants and simultaneously TPA-treated control-transfectants were compared to the appropriate TPA-treated VCP/p97 siRNA transfectants to exclude the genes affected by the transfection itself. Thereafter, the regulated genes of untreated and TPA-treated populations were compared in order to evaluate those transcripts affected by VCP/p97 during the differentiation process. Potential off-target effects have been addressed by separate analysis of the two distinct VCP/p97 siRNAs in U937 and TPA-treated U937 cells, respectively. A representative panel of resulting down-modulated (C) and up-regulated (D) VCP/p97-affected genes during differentiation are demonstrated.