Cells were seeded and grown to 50–70% confluence, and then transfected using jetPEI. For the gagAkt or PCMV6 plasmids, 1 or 2 mg of DNA/p60 was diluted in 48 ml of 150 mM NaCl. Separately, 4 ml of jetPEI was diluted in 46 ml of 150 mM NaCl and mixed with the DNA solution. This mixture was vortexed, incubated for 20 min at room temperature, and then added to the cells.

Thrombospondin-1 is a matricellular protein with potent antitumour activities, the levels of which determine the fate of many different tumours, including renal carcinomas. However, the factors that regulate this protein remain unclear. In renal carcinomas, hypoxic conditions enhance the expression of angiogenic factors that help adapt tumour cells to their hostile environment. Therefore, we hypothesized that anti-angiogenic factors should correspondingly be dampened. Indeed, we found that hypoxia decreased the thrombospondin-1 protein in several clear cell renal carcinoma cell lines (ccRCC), although no transcriptional regulation was observed. Furthermore, we proved that hypoxia stimulates multiple signals that independently contribute to diminish thrombospondin-1 in ccRCC, which include a decrease in the activity of oxygen-dependent prolylhydroxylases (PHDs) and activation of the PI3K/Akt signalling pathway. In addition, thrombospondin-1 regulation in hypoxia proved to be important for ccRCC cell migration and invasion.