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. 2017 Aug 31;16(11):1938–1957. doi: 10.1074/mcp.M116.064949

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© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

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Fig. 7. — Vorinostat enhances the expression and activity of lysosomal acid lipase (LIPA) in WT and NPC1^I1061T fibroblasts. A, Human WT and NPC1^I1061T fibroblasts were treated with DMSO and Vorinostat. Cell lysates were resolved using SDS-PAGE and Western blotted with LIPA specific antibody. GADPH was used as the loading control. Bar graph represents the relative amount of LIPA protein normalized to the loading control protein from three independent experiments. ***p < 0.001, *p < 0.05. B, DMSO- or Vorinostat-treated WT and NPC1^I1061T fibroblasts were treated with endo H as described in Methods. Endo H treated samples were resolved in 10% SDS-PAGE and Western blotted with LIPA specific antibody. Tubulin was used as loading control. Graph shows the quantification of endo H sensitive (white) or endo H resistant (black) form of LIPA proteins relative to total protein in each lane from two independent experiments. ***p < 0.001, *p < 0.05. C, Effect of Vorinostat on LIPA trafficking in HeLa-shNPC1 (npc1−/−) cells stably expressing either WT or I1061T NPC1. Both WT and I1061T HeLa cells were treated with DMSO or Vorinostat (10 μm) for 72 h; cell lysates were incubated in the presence (+) or absence (-) of endo H. Samples were separated using 10% SDS-PAGE and Western blotted using a LIPA specific antibody. D, Effect of Vorinostat on LIPA trafficking in HeLa-shNPC1 (npc1−/−) cells was analyzed using endo H digestion as described above followed by SDS-PAGE and Western blotting. Graph represents the quantification of endo H sensitive (white) or endo H resistant (black) form of LIPA proteins relative to total protein in each lane. E, Various concentrations of cell lysates from WT and NPC1^I1061T fibroblasts were immunoblotted to LIPA specific antibody (Top) and specific activity of LIPA (bottom) was performed as described in the Methods. F–G, Specific activity of LIPA found in DMSO- or Vorinostat-treated WT and NPC1^I1061T fibroblasts. WT and I1061T fibroblasts were treated with DMSO or Vorinostat (10 μm) for 72 h, followed by measurement of LIPA specific activity as described in the Methods. H–I, Specific activity of LIPA in the presence of DMSO or Orlistat (40 μm), an LIPA inhibitor for 72 h from WT and NPC1^I1061T fibroblasts. Cell lysates were obtained from drug-treated WT or NPC1^I1061T fibroblasts and the specific activity of LIPA were measured as described in the Methods. ***p < 0.001, **p < 0.01 and *p < 0.05. Graph shows the percent of LIPA enzymatic activity from three independent experiments.

Fig. 7. — Vorinostat enhances the expression and activity of lysosomal acid lipase (LIPA) in WT and NPC1^I1061T fibroblasts. A, Human WT and NPC1^I1061T fibroblasts were treated with DMSO and Vorinostat. Cell lysates were resolved using SDS-PAGE and Western blotted with LIPA specific antibody. GADPH was used as the loading control. Bar graph represents the relative amount of LIPA protein normalized to the loading control protein from three independent experiments. ***p < 0.001, *p < 0.05. B, DMSO- or Vorinostat-treated WT and NPC1^I1061T fibroblasts were treated with endo H as described in Methods. Endo H treated samples were resolved in 10% SDS-PAGE and Western blotted with LIPA specific antibody. Tubulin was used as loading control. Graph shows the quantification of endo H sensitive (white) or endo H resistant (black) form of LIPA proteins relative to total protein in each lane from two independent experiments. ***p < 0.001, *p < 0.05. C, Effect of Vorinostat on LIPA trafficking in HeLa-shNPC1 (npc1−/−) cells stably expressing either WT or I1061T NPC1. Both WT and I1061T HeLa cells were treated with DMSO or Vorinostat (10 μm) for 72 h; cell lysates were incubated in the presence (+) or absence (-) of endo H. Samples were separated using 10% SDS-PAGE and Western blotted using a LIPA specific antibody. D, Effect of Vorinostat on LIPA trafficking in HeLa-shNPC1 (npc1−/−) cells was analyzed using endo H digestion as described above followed by SDS-PAGE and Western blotting. Graph represents the quantification of endo H sensitive (white) or endo H resistant (black) form of LIPA proteins relative to total protein in each lane. E, Various concentrations of cell lysates from WT and NPC1^I1061T fibroblasts were immunoblotted to LIPA specific antibody (Top) and specific activity of LIPA (bottom) was performed as described in the Methods. F–G, Specific activity of LIPA found in DMSO- or Vorinostat-treated WT and NPC1^I1061T fibroblasts. WT and I1061T fibroblasts were treated with DMSO or Vorinostat (10 μm) for 72 h, followed by measurement of LIPA specific activity as described in the Methods. H–I, Specific activity of LIPA in the presence of DMSO or Orlistat (40 μm), an LIPA inhibitor for 72 h from WT and NPC1^I1061T fibroblasts. Cell lysates were obtained from drug-treated WT or NPC1^I1061T fibroblasts and the specific activity of LIPA were measured as described in the Methods. ***p < 0.001, **p < 0.01 and *p < 0.05. Graph shows the percent of LIPA enzymatic activity from three independent experiments.