ACF induced apoptosis and suppressed c-Myc expression and stem cell potential in CML cells. Cells were treated with PBS or 5 µM ACF or 1 µM IM and incubated at 0.1% O₂ for the indicated times (primary culture; LC1). (A) Viable cells were counted at the indicated times of LC1. Dashed line indicates the number of plated cells. Values represent mean ± S.D. of data obtained from 3 independent experiments, each carried out in triplicate; *P ≤ .05, **P ≤ .01. (B) 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. (C) Cells were stained with annexin V–APC and analyzed by flow cytometry. Values represent mean ± S.D. of data obtained from 3 independent experiments, each carried out in triplicate; *P ≤ .05. (D) 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 and densitometric analysis of data from 4 independent experiments are shown; values represent mean ± S.D.; vs PBS: *P ≤ .05, **P ≤ .01; vs IM: #P ≤ .05. (E) Cells were transferred from day 7 LC1 to drug-free normoxic secondary cultures (LC2) to determine the maintenance of stem cell potential in LC1 via the counting of viable cells at the indicated times of incubation in LC2. Values represent mean ± S.D. of 3 independent experiments. (F) K562 cells stably transfected with shRNA against HIF-1α (shHIF-1α) or control shRNA were incubated in low-oxygen LC1 for 7 days and then transferred to normoxic LC2, to count viable cells at the indicated times of incubation in LC2. Values represent mean ± S.D. of 3 independent experiments. (F, inset) Cells from time 0 LC1 were lysed and subjected to immunoblotting with the indicated antibodies. ERK1/2 was used to verify equalization of protein loading. Migration of molecular weight markers is indicated on the left (in kilodaltons). (G) Cells were treated with PBS or 5 µM ACF from day 6 to day 9 of incubation in low-oxygen LC1 and then transferred to drug-free normoxic LC2 to count viable cells. Data, relative to the peak of LC2 repopulation (day 21), are expressed as fraction of the value obtained for PBS. Values represent mean ± S.D. of data obtained from 4 independent experiments; *P ≤ .05, **P ≤ .01.