Fig. 2. Cholesterol binding stabilizes VCAM-1.

(a) Western blots for VCAM-1 and His-ALOD4 in HUVECs cultured in media containing either 10% FBS or 1% LPDS with simvastatin for 16 h before stimulation with LPS (100 ng/ml) for 0–24 h. (b and c) VCAM-1 western blots in cells stably overexpressing either human VCAM-1 or VCAM-1(∆TMD) and cultured in media containing 10% FBS, 1% LPDS with simvastatin and mevalonate overnight, or LPDS with simvastatin and mevalonate overnight before addition of increasing concentrations of MβCD-cholesterol for 2 h. (d and e) VCAM-1 western blots in HUVECs stably expressing either FLAG-VCAM-1 or FLAG-VCAM-1(Y694A) cultured in media containing 10% FBS (−), 1% LPDS with simvastatin for 8 h, or 1% LPDS with simvastatin plus 100 μM MβCD-cholesterol for 2 h. (f) Western blots for VCAM-1 in HUVECs cultured in media containing 10% FBS and stimulated with TNFα for 12 h before being placed in media containing simvastatin and either 1% LPDS, 3% FBS or 10% FBS for a further 12 h. The top rows show the NP-40 resistant portion of cells while the bottom rows show the NP-40 soluble portion of cells. (g) VCAM-1 western blots showing the effects of proteasome inhibition with MG-132 in HUVECs stably overexpressing FLAG-VCAM-1 and cultured in 10% FBS, 1% LPDS with simvastatin overnight or LPDS with simvastatin plus 100 μM MβCD-cholesterol for 1 h. (h and i) HUVECs were treated with either LPS or TNFα for 36 h before being incubated with chloroquine (10 μM) or MG132 (10 μM) for a further 12 hours. VCAM-1 and MCL-1 (positive control for MG-132) were assessed by western blot. (j) HUVECs expressing FLAG-VCAM-1 were cultured in 1% LPDS with simvastatin overnight before being switched to media containing MG132 (10 μM) in 1% LPDS with simvastatin or 1% LPDS with simvastatin plus 20% FBS for 4 h. FLAG was immunoprecipitated before VCAM-1 and ubiquitin were assessed by western blot. (k and l) HUVECs expressing FLAG-VCAM-1 or FLAG-VCAM-1(K736A) were switched from media containing 10% FBS to media containing 1% LPDS with simvastatin for 12 h to assess their rate of degradation in cholesterol deplete conditions by western blotting. (m) Western blot for VCAM-1 in the lungs of male WT mice that received i.v. infusions of either saline or LDL for 6 h (n = 7 per group). (n) Quantification of VCAM-1 relative to Ve-cadherin measured by western blot in the lungs of WT mice after i.v. infusions of LDL or saline for 6 h. (o) mRNA levels of Vcam1 relative to 36b4 in the lungs of male WT mice that received i.v. infusions of either saline or LDL for 6 h (n=7 saline and 8 LDL). Data are represented as mean ± SEM with individual mice represented by dots.