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Fig. 3. Oxygen-independent modulation of HIF expression during TS cell differentiation. (A) Levels of mRNAs encoding Arnt, HIF1 and HIF2 were assayed by Northern blot hybridization after differentiation under standard tissue culture conditions for 1 week. (B) Protein levels of the HIFs and related factors were assayed by immunoblot. ARNT protein was solely detected in the nuclei of wild-type TS cells and levels did not change with differentiation. In normoxia (20% O₂), HIF1 expression was strongly induced in wild-type but not mutant TS cells. With regard to HIF2, high cytoplasmic expression was observed only after differentiation of Arnt-null TS cells, with comparable levels of nuclear expression in both wild-type and mutant cells after FGF4 and heparin withdrawal. pVHL levels, in both the cytoplasmic and nuclear fractions, were induced during differentiation. HSP90ß expression, which was confined to the nucleus, did not change significantly during either wild-type or mutant TS cell differentiation. (C) The effects of hypoxia (2% O₂) on ARNT, HIF1 and HSP90ß expression in undifferentiated wild-type and mutant TS cells mirrored the effects of differentiation. (D) As expected, mRNA levels of the HIF1 target genes VEGF and GLUT1 were upregulated when wild type, but not mutant TS cells were cultured under hypoxic conditions. PGK1, which was upregulated by hypoxia in wild-type TS cells, was expressed only at low levels in the Arnt-null mutants.

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