Fig. 6.

Effect of downregulation of PAP7 by siRNA on expression of genes involved in cellular iron metabolism. As detailed in the materials and methods, K562 cells at various times after transfection with small interfering (si)PAP7–3 or with vector alone or mock transfection were lysed and protein and mRNA levels were determined. There were no significant differences between mock and vector alone transfections. Hence, we used vector alone as a reference to validate the effects of siPAP7–3 vector on the mRNA and protein expression levels of PAP7 (■), DMT1 total (♦) (Western blot only), DMT1 (IRE) (●), DMT1 (non-IRE) (▲), TfR1 (△), and H-ferritin (□), which were measured either by real-time PCR (A) or by Western blot analysis (B), respectively. Shown are the ratios for each day examined after transfection with siPAP7–3 to vector alone for the mRNA (A) or the signal intensity from scans of the Western blots (mean levels ± SE for 3 experiments). An unpaired t-test was applied to the results: *P < 0.05, significant difference from vector alone transfected cells.