HIF-1α was expressed in CML cells and ACF inhibited the increase of HIF-1α target genes in low oxygen. (A) Primary (CML case 4) or CML cell lines were lysed and total cell lysates subjected to immunoblotting with the indicated antibodies. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a loading control. Migration of molecular weight markers is indicated on the left (in kilodaltons). One representative experiment of 3 is shown. (B-C) Cells were incubated at the indicated oxygen concentrations for 3 days. (B) Nuclear lysates were subjected to immunoblotting with the indicated antibodies. Fibrillarin was used to verify the equalization of protein loading. Migration of molecular weight markers is indicated on the left (in kilodaltons). One representative experiment of 3 is shown. (C) Total cell lysates were subjected to immunoblotting with the indicated antibodies. GAPDH was used to verify equalization of protein loading. Migration of molecular weight markers is indicated on the left (in kilodaltons). One representative experiment of 3 is shown. (D) Cells were incubated for 2 days at 0.1% O2, in the presence of 5 µM ACF or 50 µM YC1, or their solvents (PBS or DMSO, respectively). CAIX, VEGF, or HIF-1α mRNA were measured by q-PCR. Data were normalized with respect to β-actin and expressed as fold-change with respect to the values obtained for time 0 (t0) cells. Values represent mean ± standard deviation (S.D.) of 3 independent experiments, each carried out in triplicate; vs t0: *P ≤ .05, **P ≤ .01; vs control (PBS or DMSO) 0.1% O2: #P ≤ .05, ##P ≤ .01; either comparison: n.s., not significant.
