Fig. 5. PAI-1 sequesters tPA away from apoB, leading to increased VLDL assembly in hepatocytes.

(A) Human primary hepatocytes lysates (input) and anti–PAI-1 immunoprecipitates (IP: PAI-1) were assayed for tPA and PAI-1 by immunoblot. (B) A proximity ligation assay was used to measure tPA–PAI-1 interaction in human primary hepatocytes. Scale bars, 10 μm. (C) Human primary hepatocytes were treated for 6 hours with 0.4 mM oleate complexed with fatty acid–free BSA (oleate group) or fatty acid–free BSA alone (vehicle control). Cell lysates were assayed for tPA by immunoblot using the Jess Simple Western system. (D) Human primary hepatocytes were treated with 0.4 mM oleate complexed with fatty acid–free BSA (oleate group) or fatty acid–free BSA alone (vehicle control). Cell lysates were assayed for tPA–PA-1 complex and PAI-1–free tPA concentrations by ELISA. (E) Human primary hepatocytes were treated with siRNA against PAI-1 mRNA (si-PAI1) or scrambled RNA and then incubated in medium containing either 0.4 mM oleate complexed with fatty acid–free BSA (oleate group) or fatty acid–free BSA alone (vehicle control). Cell lysates were assayed for PAI-1–free tPA concentration by ELISA. (F) Human primary hepatocytes were treated with siRNA against PAI-1 mRNA (si-PAI1) or scrambled RNA and then incubated in medium containing either 0.4 mM oleate complexed with fatty acid–free BSA (oleate group) or fatty acid–free BSA alone (vehicle control). apoB secretion was measured by [³H]-labeling, as in Fig. 2. (G) Human primary hepatocytes were treated with siRNA against tPA mRNA (si-tPA) or against PAI-1 mRNA (si-PAI1) or scrambled RNA. apoB secretion was measured by [³H]-labeling, as in Fig. 2. (H) A solid-phase binding assay was used to measure the interaction between immobilized LDL and recombinant human tPA or tPA–PAI-1 complex. (I) The effect of recombinant human tPA and tPA–PAI-1 complex on lipid transfer from donor vesicles to human LDL was assayed. (J) Normal chow diet–fed WT mice had their food withdrawn for 5 hours and were then euthanized at 0, 1, 2, and 6 hours after oral gavage with olive oil. Liver lysates were assayed for PAI-1–free tPA concentration by ELISA. Data are shown as means ± SEMs; *P < 0.05 by one-way ANOVA followed by Dunnett’s test [(D) to (G), (I), and (J)]. n.s., not significant (P ≥ 0.05).