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. 2008 Apr 18;283(16):10967–10977. doi: 10.1074/jbc.M709741200

FIGURE 4.

FIGURE 4.

Knockdown and transfection experiments in H9c2 cells using siRNA to sequences in the open-reading frame of SDHa mRNA. A, RT-PCR for SDHa normalized to 18 S rRNA. Lanes 1 and 2 shows SDHa mRNA expression in control cells. Lanes 3, 4, lanes 5, 6, and lanes 7, 8 show effects of SDHa siRNA (80 μm) on mRNA expression at 24, 48, and 72 h, respectively. The histogram represents SDHa induction by real time PT-PCR (n = 3; *, p < 0.05 compared with control). B, Western blotting for mitochondrial succinate-ubiquinol oxidoreductase (SDH) subunit A and B proteins, the two functionally active subunits of Complex II. The blot shows controls (lanes 1 and 2) and cells transfected with siRNA to the SDHa subunit (lanes 3-8). Porin is used as a loading comparison. Lanes 3-6 show SDHa siRNA transiently inhibits SDHa but not SDHb protein expression. C, in-gel quantification of Complex II enzyme activity in control and SDHa-silenced H9c2 cells at two concentrations of SDHa siRNA (40 and 80 μm). Native gels were incubated in reaction buffer containing 10 mm succinate and developed with nitroblue tetrazolium. D, nuclear HIF-1α and NRF-1 in SDHa-silenced cells. SDHa silencing increases HIF-1α but not NRF-1 nuclear protein accumulation. Cells were transfected with either scRNA (controls) or siRNA to SDHa subunit. After transfection, nuclear HIF1α and NRF-1 were analyzed by Western blot. Tubulin is shown for a loading comparison. E, PHD activity in SDHa-silenced cells. PHD activity was assessed by detection of hydroxylated proline 564 of HIF-1α (Hyp-564) by immunoprecipitation followed by Western blot analysis. Proteasomal degradation was inhibited with 5 μm MG132 (2-h incubation). Aerobic H9c2 cells were incubated with 1 mm DMOG for 2 h to inhibit PHD. Cells were transfected with either scRNA or siRNA for the SDHa subunit. The last two lanes show the effect of siNRF-1. Total HIF-1α is shown for comparison. F, SDHa transfection and NRF-1 knockdown in primary rat cardiomyocytes. Left shows Western blot analysis of nuclear HIF-1α and NRF-1 relative to tubulin. SDHa protein is shown relative to β-actin. Lane 1, control cells. Lane 2, SDHa overexpression and SDHa, NRF-1, and HIF-1α protein expression at 48 h. Lane 3, SDHa, NRF-1, and HIF-1α expression 48 h after siNRF-1. Lane 4, co-transfection of SDHa-pCMV6-XL5 plasmid with siNRF-1 (low NRF-1 and high SDHa expression) prevents nuclear HIF-1α protein accumulation. The histogram (right) shows HIF-1α nuclear protein relative to tubulin by densitometry. Data are means of n = 4 (*, p < 0.05 compared with control).