T2A inhibited HIF-1α expression through the inhibition of protein synthesis rather than the promotion of HIF-1α degradation.

<p>(A) The MDA-MB-231 cells were cultured under normoxic or hypoxic conditions for 8 hours, then pretreated without or with 20 μM T2A for 30 minutes, followed by treatment without or with 20 μM cycloheximide (CHX) for various intervals as indicated. Upper panel: after treatment, whole cell extracts were analyzed by Western blot using antibodies against HIF-1α and β-actin. Lower panel: quantification of the HIF-1α by densitometry following normalization to the β-actin level. The HIF-1α protein levels in untreated or T2A-treated cells were arbitrarily given the value of 100%. (B) Cells were labeled with [³⁵S]methionine as described in the Methods section and chased for the indicated periods (h) in the presence or absence of T2A under normoxic or hypoxic conditions. Upper panel: equal amounts of proteins from each cell lysate were subjected to immunoprecipitation and then separated in SDS-PAGE and examined by autoradiography following normalization to the control. Lower panel: quantification of the autoradiographic HIF-1α signal by densitometry. (C) Cells were pretreated with either vehicle or 20 μM T2A for 16 hours and then subjected to hypoxia, or remained in normoxia for an additional 8 hours. Subsequently, cells were labeled with [³⁵S]methionine in the presence or absence of 20 μM T2A for the indicated time. Upper panel: Equal amounts of proteins were subjected to immunoprecipitation and then separated in SDS-PAGE and examined by autoradiography. Lower panel: quantification of the autoradiographic HIF-1α signal by densitometry. (D) Cells were pretreated with the proteasome inhibitor MG132 (10 μM) for 2 hours, followed by T2A treatment (20 μM) under normoxic or hypoxic conditions as described above. Whole cell extracts were analyzed by Western blot using antibodies against HIF-1α and β-actin.