Figure 5. Gene silencing of MT1-MMP antagonizes the effects of hypoxia on 3BP2 gene expression.

MSC were transiently transfected with scrambled sequences (Mock, white bars) or MT1-MMP siRNA (black bars) as described in the Methods section. Cells were then cultured under normoxic or hypoxic culture conditions. (A) Immunoblotting of MT1-MMP protein expression was evaluated in cell lysates from siRNA experiments. Total RNA was extracted, and qRT-PCR was used to assess 3BP2, HIF-1α and MT1-MMP. Apoptotic cell death was assessed using the fluorometric caspase-3 activity assay as described in the Methods section. (B) 3BP2 and HIF-1α gene expression were assessed by qRT-PCR in Mock-transfected (open circles) and in siMT1-MMP-transfected (closed circles) cells that were subsequently cultured under hypoxic conditions. (C) MSC were transiently transfected with either scrambled sequences or MT1-MMP siRNA as described in the Methods section. Cells were then cultured under normoxic or hypoxic culture conditions, cell lysates were isolated, western blotting and immunodetection were performed with anti-3BP2 and anti-GAPDH antibodies. Values in (A) and (B) are means of two independent experiments, each performed in triplicates (*p<0.05 versus mock control in (A) or mock at time = 0 hr hypoxia in (B)); Bars, ±SD.