FIGURE 7. PIAS3 knockdown decreases basal expression and oxidative stress-mediated induction of ferritin H.

A,1 × 10⁷ K562 cells were transfected with 100 pmol (final, 1 μm) of nontargeting siRNA (siControl) or PIAS3-targeting siRNA (siPIAS3, M-004164-05; Dharmacon). After 40 h of transfection, the cells were treated with 50 μm t-BHQ or 50 μm hemin for 8 h. 50 μg of each cell lysate was subjected to Western blotting with antibodies for PIAS3, ferritin H, and β-actin. The experiment was repeated three times, and the representative results of Western blots are shown. B,5 × 10⁶ K562 cells were transfected with 100 pmol (final, 1 μm) of no siRNA (no siRNA), nontargeting siRNA (siControl) or PIAS3 siRNA (siPIAS3, J-004164-12; Dharmacon). After 48 h of transfection, the cells were treated with 10 μm t-BHQ or 10 μm hemin for 24 h. 5 μg of total RNA was subjected to Northern blotting probed with ³²P-labeled ferritin H cDNA. Ethidium bromide staining of total RNA is shown below to verify equal loading and integrity of RNA. The positions of 28 and 18 S ribosomal RNA are indicated. 50 μg of whole cell lysates were analyzed for expression of PIAS3 and β-actin proteins by Western blotting.