Fig 1. Sustained mTORC1, but not mTORC2, inhibition activates Akt.

HUVECs were treated by rapamycin, two different small interfering RNAs against rictor (siRict¹ and siRict²) or raptor (siRapt), or non-silencing siRNA (siNS), then stimulated with 20 ng/mL VEGF as indicated. Akt phosphorylation and S6K phosphorylation were evaluated as described in Materials and Methods. A) A representative Western blot of the effect of Rapamycin pretreatment for 1 hour on VEGF-stimulated Akt and S6 kinase phosphorylation in HUVECs. B) Quantitation of phospho-S6K. C) Quantitation of phospho-Akt (n = 5 independent experiments, *P<0.05 by ANOVA). The effect of rapamycin treatment of HUVEC for 24 hours. D) A representative Western blot of HUVEC phospho-Akt, total-Akt1 and tubulin over a range of concentration of rapamycin exposure. E) Quantitation of phospho-Akt (n = 5 independent experiments, *P<0.05 by ANOVA). The effect of mTORC1 versus mTORC2 disruption on Akt signaling in EC. F) A representative Western blot of HUVEC rictor, raptor, phospho-Forkhead box protein O1/3 (P-FOXO1/3), phospho-Akt, total Akt1, phospho-S6K, total S6K, and actin after treatment with siRapt, siRict or siNS. G) Quantitation of phospho-Akt. H) Quantitation of phospho-S6K (n = 3 independent experiments, *P<0.05 by ANOVA).