Figure 4.

HIF-1α under NO treatment. HIF-1α expression level (A) and HIF-1α protein level (B) in WT MDA-MB-468 monolayer cells treated with 500 µM GNSO for up to 8 h under normoxia (21% O2) and for protein analysis also hypoxia (1% O2), where cells had already been under HOX for 24 h before addition of GNSO. HIF-1α expression was not altered after GNSO addition. Expression normalized to ribosomal protein S16 (rP); mean +/- sd; n = 4 - 6 in 3 independent experiments. Under normoxia, HIF-1α protein significantly increased 2 h after addition of GNSO while reaching basal levels again after 6 h. Under hypoxia, overall HIF-1α significantly increased compared to normoxic conditions whereas GNSO addition led to no additional changes of HIF-1α level. HIF-1α (120 kDa) normalized to ß-ACTIN (42 kDa) as house-keeping gene; mean +/- sd; n = 12 in 4 independent experiments (NOX) and n = 4 in 2 independent experiments (HOX). (C, D) Overall HIF-1α and hydroxylated HIF-1α-OH protein levels of WT and MB KD MDA-MB-468 monolayer cells treated with 500 µM GNSO for up to 6 h under normoxia. 3 h after addition of GNSO, both WT and KD cells showed their highest overall HIF-1α level, with a higher tendency of KD cells compared to WT cells. n = 6-10 in 5 independent experiments (DMOG: n = 2). In contrast, hydroxylated HIF-1α-OH showed an increase only in WT cells whereas in KD cells the level remained constant and significantly lower at 4 h time-point. Addition of dimethyloxaloylglycine (DMOG), a prolyl hydroxylase-inhibitor, was used as a positive control of HIF-1α stabilization. HIF-1α/HIF-1α-OH (120 kDa) normalized to ß-ACTIN (42 kDa) as house-keeping gene; mean +/- sd; n = 4 in 2 independent experiments. *p < 0.05 as indicated.