Fig. 1.

a Schematic detailing the inducible system used in our novel cell line model. A mutant form of HIF1-alpha (mHIF) which is stable in normoxia is fused to a YFP-tagged destabilising domain. The fusion is readily transcribed and translated but is unstable and broken down in the absence of Trimethoprim (TMP). When TMP is added the fusion is stabilised and expressed. b Stabilisation and expression following addition of 10 μM TMP was assessed by Western blot of nuclear extracts. Western blots were performed on single membranes cut in two with upper section labelled for mHIF and lower section labelled for Lamin B1. c To activate downstream signalling HIF1-alpha must interact with HIF1-beta to translocate into the nucleus. Successful nuclear translocation was verified by immunocytochemistry. Scale bar equal to 25 μm. d qRT-PCR was performed on control (YFP-DD) and inducible (mHIF-YFP-DD) cells in the presence or absence of 10 μM TMP. Gene expression was seen to change in the inducible cells suggesting HIF1-alpha activation of signalling. e Expression of mHIF-YFP-DD was measured over time following addition of TMP (green bars) and following wash out of TMP (red bars) in both MCF7 and 231 lines. Significantly increased expression was seen in both lines within 1 h and the signal had returned to normal after 8 h. f Expression of mHIF-YFP-DD was measured over time following addition of a single dose of TMP at Day 0. Expression remained elevated over 4 days