Figure 6.
NCOA4 protein induction by DFO is modulated by HIF knockdown. (A) Immunoblotting analyses of NCOA4, HIF-1α, HIF-2α, and β-actin in protein lysates harvested from transfected Hep3B cells either before DFO treatment (−) or after 12, 18, or 24 hours of DFO treatment. For panel A, Hep3B cells, which had been grown in CM supplemented with 50 μM FAC (CM+FAC), were seeded at 2.5 × 105 cells per well in 6-well plates and reverse transfected with either scrambled siRNA alone (10 nM) or with both HIF1A siRNA and HIF2A siRNA (each at 5 nM) in CM+FAC without antibiotics. Twenty-four hours later, the media were replaced with antibiotic-free CM supplemented with 100 μM DFO. Proteins were harvested from each siRNA group at 4 timepoints: immediately before addition of DFO-supplemented CM (0 hr) and at 12, 18, and 24 hours after addition of DFO-supplemented media, as diagramed in supplemental Figure 11. n = 2 per group. (B) Immunoblotting analyses of NCOA4, HIF-1α, HIF-2α, and β-actin in protein lysates harvested from Hep3B cells that were or were not treated with DFO, after transfection with the indicated siRNAs. The mean band density of NCOA4 relative to β-actin from Hep3B cells transfected with scrambled siRNA and not treated with DFO was normalized to 1. (C) Immunoblotting analyses of NCOA4, HIF-1α, HIF-2α, and β-actin in protein lysates harvested from Hep3B cells that were treated with DFO, after transfection with the indicated siRNAs. To facilitate relative comparisons between data in panels B and C, the data in panel C have been normalized by setting the mean NCOA4-to-β-actin band density ratio from the DFO-treated scrambled siRNA group (gray hatched bars in panel C) to its normalized mean value from panel B (gray hatched bars in panel B). For panels B and C, Hep3B cells (grown in CM supplemented with 50 μM FAC) were seeded at 2.5 × 105 cells per well in 6-well plates and reverse transfected with scrambled siRNA, HIF1A siRNA, and/or HIF2A siRNA in the same combinations and concentrations used in Figure 5. Twenty-four hours after transfection, media was replaced with CM without antibiotics supplemented with 100 μM DFO (+DFO) or with CM without antibiotics (−DFO). Proteins were harvested 12 hours after the change in media. n = 4 per group. For both bar graphs, data represent mean ± SD. **P < .01, ***P < .001, and ****P < .0001 by 2-way (panel B) or 1-way ANOVA (panel C) with Tukey’s post hoc test. Numbers on the right indicate the position of molecular weight markers in kiloDaltons.

NCOA4 protein induction by DFO is modulated by HIF knockdown. (A) Immunoblotting analyses of NCOA4, HIF-1α, HIF-2α, and β-actin in protein lysates harvested from transfected Hep3B cells either before DFO treatment (−) or after 12, 18, or 24 hours of DFO treatment. For panel A, Hep3B cells, which had been grown in CM supplemented with 50 μM FAC (CM+FAC), were seeded at 2.5 × 105 cells per well in 6-well plates and reverse transfected with either scrambled siRNA alone (10 nM) or with both HIF1A siRNA and HIF2A siRNA (each at 5 nM) in CM+FAC without antibiotics. Twenty-four hours later, the media were replaced with antibiotic-free CM supplemented with 100 μM DFO. Proteins were harvested from each siRNA group at 4 timepoints: immediately before addition of DFO-supplemented CM (0 hr) and at 12, 18, and 24 hours after addition of DFO-supplemented media, as diagramed in supplemental Figure 11. n = 2 per group. (B) Immunoblotting analyses of NCOA4, HIF-1α, HIF-2α, and β-actin in protein lysates harvested from Hep3B cells that were or were not treated with DFO, after transfection with the indicated siRNAs. The mean band density of NCOA4 relative to β-actin from Hep3B cells transfected with scrambled siRNA and not treated with DFO was normalized to 1. (C) Immunoblotting analyses of NCOA4, HIF-1α, HIF-2α, and β-actin in protein lysates harvested from Hep3B cells that were treated with DFO, after transfection with the indicated siRNAs. To facilitate relative comparisons between data in panels B and C, the data in panel C have been normalized by setting the mean NCOA4-to-β-actin band density ratio from the DFO-treated scrambled siRNA group (gray hatched bars in panel C) to its normalized mean value from panel B (gray hatched bars in panel B). For panels B and C, Hep3B cells (grown in CM supplemented with 50 μM FAC) were seeded at 2.5 × 105 cells per well in 6-well plates and reverse transfected with scrambled siRNA, HIF1A siRNA, and/or HIF2A siRNA in the same combinations and concentrations used in Figure 5. Twenty-four hours after transfection, media was replaced with CM without antibiotics supplemented with 100 μM DFO (+DFO) or with CM without antibiotics (−DFO). Proteins were harvested 12 hours after the change in media. n = 4 per group. For both bar graphs, data represent mean ± SD. **P < .01, ***P < .001, and ****P < .0001 by 2-way (panel B) or 1-way ANOVA (panel C) with Tukey’s post hoc test. Numbers on the right indicate the position of molecular weight markers in kiloDaltons.

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