Alteration of the VCP/p97 expression during differentiation and retrodifferentiation. A. U937 cells were treated initially with 10 nM TPA and then cultured in the absence of TPA until retrodifferentiation. At the time points indicated, cell lysates were separated by 10% SDS-PAGE followed by Western blot analysis with anti-VCP/p97, anti-P-tyr, anti-p21Cip1/Waf1/sdi-1 and anti-vimentin antibody. The tyrosine phosphorylation pattern (p-tyr) is demonstrated by the complete range from 250-20 kDa. Staining with β-actin was used as a loading control. Densitometric analysis and normalization to β-actin was performed using the ImageJ software (NIH, Bethesda, MD, USA). B. Quantitative RT-PCR analysis of VCP/p97 mRNA levels during differentiation and retrodifferentiation. Following total RNA isolation of undifferentiated U937 control cells (U937), differentiated populations (3d until 18d) and retrodifferentiated U937 cells (Retro), the amplification was performed in the LightCycler 2.0 System using the LightCycler Software 3.5. Results represent means ± SD from three independent experiments.