Rapamycin reduces VCAM-1 expression by inhibiting mTORC2. (A) TNF-activated control- or rapa-ECs were subjected to adhesion assays using human memory T cells. (B) TNF-activated control- and rapa-ECs were analyzed by FACS. (C) ECs pretreated with rapamycin were treated with TNF and assayed by FACS. Data are normalized to median fluorescence intensity (MFI) of staining in control-ECs. (D) VCAM-1 mRNA expression in control- or rapa-ECs stimulated with TNF. (E) Control- or rapa-ECs transduced with the indicated luciferase reporters were stimulated with TNF and assayed for luciferase expression. Values are normalized to expression in control-ECs (no TNF, no rapamycin). (F) Quantification of nascent VCAM-1 and E-selectin mRNA expression in control- and rapa-ECs stimulated with TNF. (G) TNF-activated GFP- or mTOR-knockdown ECs were assayed by Western blotting. (H) TNF-activated GFP-, rictor-, or raptor-knockdown ECs were assayed by Western blotting. (I) TNF-activated GFP-, rictor-, or raptor-knockdown ECs were subjected to adhesion assay. (J) TNF-activated control or VCAM-1–overexpressing ECs were transduced with GFP or rictor shRNA and subjected to adhesion assay. For G and H, similar results were seen in two independent experiments. Mean ± SEM are shown for data pooled from two (A [n = 6], C [n = 4], E [n = 6], F [n = 5], I [n = 6], and J [n = 6]) or three (B [n = 3] and D [n = 4]) independent experiments (n = total replicates). For C, E, and J, significance was determined by ANOVA with Tukey’s post-hoc test; all other data were analyzed with the Student’s t test. *, P < 0.05.