Fig. 3

CRABP-II knockout decreased lipid raft cholesterol accumulation and AKT survival signaling. (A) Comparison of cholesterol content in CRABP-II knockout cells and in wild type control Panc-1 cells. The free cholesterol (FC) and esterified cholesterol (EC) were assessed by liquid chromatography with tandem mass spectrometry (LC–MS-MS) and normalized to total protein. (B) Comparison of lipid raft cholesterol storage in wild type cells and CRABP-II knockout cells. The membrane lipid raft fractions were isolated by sucrose gradient ultracentrifuge (up panel) and the total cholesterol was assessed using cholesterol assay kit and normalized to total protein. The green bar presents the CIIKO cells pretreated with 1 mM cholesterol in media. (C, D) AKT activation assessed by western blots. Panc-1, CIIKO and cholesterol pretreated CIIKO cells were incubated with 100 nM insulin for 0, 15, 30 and 60 min, cells were immediately lysed. (E, F) Rescuing the loss of raft-cholesterol and AKT activation in CIIKO cells by re-expression of CRABP-II. (G, H) Increased raft-cholesterol content and AKT activation in gemcitabine resistant cell lines. All these experiments were repeated at least three times and the error bars present SD. **, p < 0.01