Figure 3
From: Microenvironmental Hypoxia regulates FLT3 expression and biology in AML
FLT3 regulating mechanisms by hypoxia.
(A) Representative experiment of FLT3 degradation in Ba/F3 W51 at 21% and 1% O2 after protein synthesis inhibition with CHX (50 μg/mL) showing faster and increased degradation during hypoxia (immature intracellular FLT3 receptor: 130 kDa, mature glycosylated FLT3 receptor: 155 kDa). (B) Fold change of FLT3 OD normalized to β-actin. t½ is shortened during hypoxia by 1 hour from 2.5 hours to 1.5 hours. (C) Degradation of FLT3 in Ba/F3 W51 cells remains proteasome dependent also at 1% O2, as treatment with proteasome inhibitor MG132 reverses degradation after protein synthesis blockade with CHX. Fold change of FLT3 OD for each time point is calculated compared to the 0 time point value and normalized to β-actin (D) mRNA levels for FLT3 were unaltered after exposure to 72 hours of hypoxia in Ba/F3 cells. (E) Effects of HSP90 inhibition, Hif1alpha activation and PI3-Kinase inhibition on Ba/F3 W51 cells (cells were adjusted at hypoxia for 24 hours and treated for 48 hours) FLT3 expression and FLT3 downstream pathway activation was analysed by western blot. Only PI3-K inhibition partially abrogated hypoxia-mediated down-regulation of FLT3.
