Figure 4.
Chemicals that stabilize HIF raise NCOA4 expression in human hepatoma cells. (A) TFRC mRNA expression relative to ACTB in Hep3B cells after 18-hour treatment with the indicated concentrations of DFO. (B) NCOA4 mRNA expression relative to ACTB in Hep3B cells after 18-hour treatment with the indicated concentrations of DFO. (C) NCOA4 mRNA expression relative to ACTB in Hep3B cells after 18-hour treatment with the indicated concentrations of CoCl2. (D) NCOA4 mRNA expression relative to ACTB in Hep3B cells after 12- or 18-hour treatment with 1 mM DMOG or vehicle (dimethyl sulfoxide). For panels A-D, Hep3B cells were seeded in complete growth media (CM) at 2.5 × 105 cells/well in 6-well plates. The following day, media were replaced with CM supplemented with the indicated concentration of the respective HIF-stabilizing chemical. After the treatment period indicated, RNA was harvested for quantitative reverse transcriptase-polymerase chain reaction analysis. n = 6 per group. For panels A-C, the mean mRNA ratio from cells that were not treated with the respective HIF-stabilizing chemical was normalized to 1. For panel D, the mean mRNA ratio from vehicle-treated cells at 12 hours was normalized to 1. For all bar graphs, data represent mean ± SD. (E) Immunoblotting analyses of NCOA4, TFRC, FTL, FTH, HIF-1α, and β-actin in protein lysates from Hep3B cells with or without FAC pretreatment and harvested before DFO treatment (−) or after 12 or 18 hours of DFO treatment, as outlined in supplemental Figure 10A. Hep3B cells were seeded at 2.5 × 105 cells per well in 6-well plates in CM. After 24 hours, media were removed and replaced with either CM (−FAC) or CM supplemented with 50 μM FAC (+FAC). After an additional 24 hours, media were replaced with CM supplemented with 100 μM DFO. Proteins were harvested from the +FAC and −FAC groups at 3 timepoints: immediately before addition of DFO-supplemented CM (0 hr) and at 12 or 18 hours after addition of DFO-supplemented CM. Numbers on the right indicate the position of molecular weight markers in kiloDaltons. *P < .05 and ****P < .0001 by 1-way ANOVA (panels A-C) or 2-way ANOVA (panel D) with Tukey’s post hoc test.

Chemicals that stabilize HIF raise NCOA4 expression in human hepatoma cells. (A) TFRC mRNA expression relative to ACTB in Hep3B cells after 18-hour treatment with the indicated concentrations of DFO. (B) NCOA4 mRNA expression relative to ACTB in Hep3B cells after 18-hour treatment with the indicated concentrations of DFO. (C) NCOA4 mRNA expression relative to ACTB in Hep3B cells after 18-hour treatment with the indicated concentrations of CoCl2. (D) NCOA4 mRNA expression relative to ACTB in Hep3B cells after 12- or 18-hour treatment with 1 mM DMOG or vehicle (dimethyl sulfoxide). For panels A-D, Hep3B cells were seeded in complete growth media (CM) at 2.5 × 105 cells/well in 6-well plates. The following day, media were replaced with CM supplemented with the indicated concentration of the respective HIF-stabilizing chemical. After the treatment period indicated, RNA was harvested for quantitative reverse transcriptase-polymerase chain reaction analysis. n = 6 per group. For panels A-C, the mean mRNA ratio from cells that were not treated with the respective HIF-stabilizing chemical was normalized to 1. For panel D, the mean mRNA ratio from vehicle-treated cells at 12 hours was normalized to 1. For all bar graphs, data represent mean ± SD. (E) Immunoblotting analyses of NCOA4, TFRC, FTL, FTH, HIF-1α, and β-actin in protein lysates from Hep3B cells with or without FAC pretreatment and harvested before DFO treatment (−) or after 12 or 18 hours of DFO treatment, as outlined in supplemental Figure 10A. Hep3B cells were seeded at 2.5 × 105 cells per well in 6-well plates in CM. After 24 hours, media were removed and replaced with either CM (−FAC) or CM supplemented with 50 μM FAC (+FAC). After an additional 24 hours, media were replaced with CM supplemented with 100 μM DFO. Proteins were harvested from the +FAC and −FAC groups at 3 timepoints: immediately before addition of DFO-supplemented CM (0 hr) and at 12 or 18 hours after addition of DFO-supplemented CM. Numbers on the right indicate the position of molecular weight markers in kiloDaltons. *P < .05 and ****P < .0001 by 1-way ANOVA (panels A-C) or 2-way ANOVA (panel D) with Tukey’s post hoc test.

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