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Files in this Data Supplement:
Fig. S1. Biosynthetic delivery of ATGL to LDs is blocked by cycloheximide. (A) ATGL immunoblot of HeLa cells incubated in the absence (−) or presence (+) of 400 µM OA and/or 150 µg/ml cycloheximide (CHX) for 3 or 5 hours as indicated. An LD fraction was used as a positive control for ATGL detection. Immunoblotting for actin shows equal protein loading in all lanes. (B) Confocal microscopy images for ATGL (green) and TIP47 (red) after treating HeLa cells with either 400 µM OA (top row) or 400 µM OA and 150 µg/ml cycloheximide (bottom row) for 3 hours. Cells were fixed with 4% paraformaldehyde and immunostained with primary and secondary antibodies as described in Materials and Methods. Scale bar: 10 µm. Note that CHX treatment prevents an OA-induced increase of ATGL levels and association with LDs (A). This is consistent with the notion that ATGL recruitment to LDs requires ongoing protein synthesis.
Fig. S2. ADRP association with LDs and membranes. (A) Fluorescence recovery after photobleaching (FRAP) analysis of GFP-GGA1 on Golgi membranes and GFP-ADRP on LDs in HeLa cells incubated for 19 hours with 200 µM oleic acid (OA). Boxed regions were bleached at time 0, then fluorescence within these regions was monitored at 2-second (GFP-GGA1) or 30-second (GFP-ADRP) intervals. (B) Fluorescence values obtained as in A were plotted as a function of time. (C) HeLa cells were treated with 400 µM OA for 3 hours (top row), 400 µM OA for 3 hours followed by 400 µM OA and 5 µg/ml BFA for an additional 3 hours (middle row), or 5 µg/ml BFA for 10 minutes then 400 µM OA and 5 µg/ml BFA for 3 hours (bottom row). Cells were immunostained with antibodies to ADRP (left column) and TIP47 (right-hand column). Scale bars: 5 µm in A; 10 µm in C.
Fig. S3. Depletion of GBF1 inhibits ADRP association with LDs. (A) HeLa cells were treated for 72 hours with siRNA against GBF1 (GBF1 knockdown, KD) or mock treated, then immunostained with antibodies to ADRP and TIP47. Scale bar: 10 µm. (B) Immunoblot analysis of GBF1, ATGL, ADRP and actin (control) levels upon GBF1 KD. HeLa cells were transfected with siRNA against GBF1 or mock transfected, incubated for 60 hours, then treated with 200 µM OA for an additional 12 hours or left untreated, as indicated. Duplicate samples are shown for each condition. GBF1 ran as a doublet in this experiment. (C,D) HeLa cells were treated with GBF1 siRNA as described in A, either without (C) or with (D) addition of 400 µM OA 3 hours prior to fixation. Cells were stained with BODIPY 493/503 and LD diameters measured. The percentage of LDs falling into the indicated size ranges is plotted. Dark-grey bars, GBF1 KD cells; light-grey bars, mock-treated cells.
Fig. S4. Components of COPI and COPII coatomer complexes are required for ADRP association with LDs. (A) HeLa cells were mock treated (top row) or transfected with siRNA oligonucleotides against β-COP (middle tow) or SEC23 (bottom row) and incubated for a total of 72 hours, with 150 µM OA added for the last 12 hours. Cells were immunostained with antibodies to ADRP and BODIPY 493/503. Scale bar: 10 µm. (B) Immunoblot analysis of β-COP levels in HeLa cells treated with siRNA oligonucleotides against β-COP. Cells were treated as in A, and samples were analyzed by SDS-PAGE and immunoblotting with antibodies to β-COP and actin (loading control). (C) Immunoblot analysis of SEC23 levels in HeLa cells treated with siRNA oligonucleotides against SEC23. Cells were treated as in A, and samples were analyzed by SDS-PAGE and immunoblotting with antibodies to SEC23 and actin (loading control). (D) Expression of SAR1-T39N does not affect the size of LDs. HeLa cells were transfected with a plasmid encoding SAR1-T39N-GFP, and after 8 hours 150 µM OA was added for an additional 14 hours. Cells were stained with the lipid dye BODIPY 493/503 and LD diameters measured.
Fig. S5. The effect of GBF1, β-COP or SEC13 depletion on the delivery of ATGL to LDs. HeLa cells were mock transfected or transfected with siRNA oligonucleotides against GBF1, β-COP or SEC23, incubated with 150 µM OA for 12 hours, immunostained with antibodies to ATGL, TIP47 and either GBF1, β-COP or SEC23 and examined by confocal microscopy, as described in Materials and Methods. Bars represent the mean±s.d. from four independent experiments in which 100 cells were scored for the presence of ATGL on LDs. *P-values for significant differences between GBF1, β-COP and control cells.
Fig. S6. The Golgi protein GM130 is not found in close proximity to LDs upon OA treatment, unlike p58, the distribution of which shifts from cytosolic punta to rings surrounding LDs. (A) HeLa cells were treated for 20 hours with 150 µM OA, then co-immunostained with antibodies to GM130 (green channel, left) and to TIP47 (red channel, middle). (B) HeLa cells were treated with OA as in A, and imaged prior to OA treatment (−OA) and after 20 hours (+OA), using antibodies to p58 (green channel, left column) and to TIP47 (red channel, middle column). Merged images are shown on the right. Scale bars: 10 µm.
Movie 1. Electron tomography of a LD. The progression through a tomogram (z-stack where each virtual slice is 1 nm thick) generated using the back projection algorithm implemented in the IMOD software. Shown is the close proximity of ER and ERES/ERGIC structures to a LD. Rendered volume is presented in Fig. 6B. Gold particles (15 nm) were used as fiducials to align the tilt series.
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