Fig. 3. tPA blocks apoB-VLDL assembly.

(A) Human primary hepatocytes were treated with siRNA against tPA mRNA (si-tPA) or scrambled RNA for 24 hours. Cell lysates (input) and anti-MTP immunoprecipitates (IP: MTP) were assayed for apoB and MTP by immunoblot. (B) Human primary hepatocytes were treated with siRNA against tPA mRNA (si-tPA) or scrambled RNA (group 1) or tPA mRNA (si-tPA) (groups 2 and 3) for 24 hours. The microsomal fraction was isolated and assayed for neutral lipid transfer activity with DMSO (groups 1 and 2) or without with CP-346086 (10 nM), an MTP inhibitor (group 3). (C) Human primary hepatocytes were transduced with a plasmid encoding tPA with C terminal HA tag (tPA-HA) or GFP for 48 hours. Cell lysates (input) and anti-MTP precipitates (IP: MTP) were assayed for apoB and MTP by immunoblot. (D) Human primary hepatocytes were transduced with a plasmid encoding tPA-HA or GFP for 48 hours. The microsomal fraction was isolated and assayed for neutral lipid transfer activity. (E) The effect of recombinant human tPA on lipid transfer from donor vesicles to human LDL was assayed. (F) Human primary hepatocytes were transduced with a plasmid encoding tPA-HA or GFP for 48 hours. Cell lysates (input) and anti-HA immunoprecipitates (IP: HA) were assayed for apoB and tPA. (G) A proximity ligation assay was used to measure apoB-tPA interaction in human primary hepatocytes. Scale bars, 10 μm. (H) Confocal microscopy immunofluorescence imaging was used to measure the subcellular localization of tPA and apoB in human primary hepatocytes. Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole. (I) Human primary hepatocytes were transduced with a plasmid encoding WT tPA (tPA-WT), an enzymatically inactive mutant of tPA (tPA-S513A), tPA with an ER retention signal sequence (tPA-KDEL), or GFP for 48 hours. apoB secretion was measured by [³H]-labeling, as in Fig. 2. (J) The effect of recombinant WT tPA (tPA-WT) or enzymatically inactive mutant of tPA (tPA-S513A) on lipid transfer from donor vesicles to human LDL was assayed. Data are shown as means ± SEMs; *P < 0.05 by two-tailed Student’s t test (D) or by one-way ANOVA followed by Dunnett’s test [(B), (E), (I), and (J)].