FIGURE 3.

Anthrax LT treatment does not affect HIF-1α gene transcription and protein stability. A, quantitative PCR analysis of HIF-1α mRNA expression in HepG2 cells cultured in medium alone (M) or supplemented with LT for 1–3 h in normoxic conditions (three left columns) or pretreated with LT or medium alone (M) for 3 h, and then cultured in hypoxic conditions for an additional 4 h (two right columns). B and C, Western blotting analysis of HIF-1α protein levels in HepG2 cells pretreated in normoxic conditions with DMSO, MG132, ALLN, and/or lactacystin (Lact) for 30 min and then incubated with or without LT for 7 h as indicated. D, turnover of HIF-1α protein in HepG2 and Hepa1c1c7 cells cultured in the presence or absence of LT in hypoxic conditions. Cells were cultured in hypoxic conditions for 4 h with or without LT during the last 2 h. Cells were subsequently treated with CHX for 0, 0.5, and 1–4 h as shown. HIF-1α protein levels were determined by Western blotting. E, quantification of HIF-1α protein levels. β-Actin was used as a loading control. The intensity of protein bands in B and C was quantified using Fluorchem Q software; relative amounts are shown below each lane. Data shown are representative of three (A–C) or two (D and E) independent experiments.