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Files in this Data Supplement:
Fig. S1. Triacsin C inhibits accumulation of lipid inside ADPH-coated CLDs. Cultures stably expressing ADPH-VSV were incubated for 20 hours in unsupplemented basal media (Control, a), or in basal media supplemented with 300 µM oleic acid (b), 5 µM triacsin C (c), or 5 µM triacsin C plus 300 µM oleic acid (d). ADPH-VSV was localized by staining with mouse anti-VSV (green), neutral lipids were identified with Nile Red staining (red), and nuclei were stained with DAPI (blue). All experiments were performed at least three times with similar results. Multiple images of each were captured in each experiment; representative images are shown. Images were originally taken at 600× magnification. Scale bar: 20 µm. These data verify that CLDs observed in our cultures require neutral lipid synthesis and that neutral lipid synthesis is needed to detect ADPH.
Fig. S2. ADPH-coated CLDs observed following MG132 treatment contain neutral lipids. Cultures stably expressing ADPH-VSV were incubated for 20 hours in unsupplemented basal media (Control, a), or in basal media supplemented with 2 µM MG132 (b), 300 µM oleic acid (c) or 3 µM MG132 plus 300 µM oleic acid (d). ADPH-VSV was localized by staining with mouse anti-VSV (green), neutral lipids were identified with Nile Red staining (red), and nuclei were stained with DAPI (blue). All experiments were performed at least three times with similar results. Multiple images of each were captured in each experiment; representative images are shown. Images were originally taken at 600× magnification. Scale bar: 20 µm. These data validate that ADPH-coated CLDs induced by MG132, or MG132 plus oleic acid, contain neutral lipids.
Fig. S3. Triacsin C inhibits MG132-induced ADPH-coated CLDs. Cultures stably expressing ADPH-VSV were incubated for 20 hours in basal media supplemented with 3 µM MG132 (a) or with 3 µM MG132 plus 5 µM triacsin C (b). ADPH-VSV was localized by staining with mouse anti-VSV (red), TIP47 was localized by staining with guinea pig anti-TIP47 (green), and nuclei were stained with DAPI (blue). All experiments were performed at least three times with similar results. Multiple images of each were captured in each experiment; representative images are shown. Images were originally taken at 600× magnification. Scale bar: 20 µm. These findings verify that ADPH-coated CLDs induced in control cells by MG132 treatment are sensitive to triacsin C, suggesting that these structures contain neutral lipids.
Fig. S4. ADPH and TIP47 localizations in saponin-permeabilized cells. Cultures stably expressing ADPH-VSV (a,b) or Δ 2,3 ADPH-VSV (c,d) were incubated for 20 hours in unsupplemented basal media (a,c) or in basal media supplemented with 300 µM oleic acid (b,d). Both versions of ADPH were localized by staining with mouse anti-VSV (red), TIP47 was localized by staining with chicken anti-TIP47 (green), and nuclei were stained with DAPI (blue). All experiments were performed at least three times with similar results. Multiple images of each were captured in each experiment; representative images are shown. Images were originally taken at 600× magnification. Scale bar: 20 µm. These results are similar to those observed for ethanol-permeabilized cells.
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