(A) Association of intact ER to LDs. Total LDs were isolated from CHO K2 cells and distributed into 12 aliquots. The aliquots were treated with trypsin at 37 °C at the concentration (%, w/v) and times indicated. After incubation, LDs were re-isolated, and proteins were extracted with chloroform:acetone (300 μl:700 μl) and analyzed by Colloidal Blue staining and Western blotting with the indicated antibodies. (B) Treatment of LDs with high concentration salt. Isolated LDs were incubated with or without 2 M NaCl in Buffer B at 4 °C for 30 min. After incubation the reaction was centrifuged at 20,000 g for 3 min. LDs layer (top) and the solution underneath (Solu) were collected respectively. LD proteins were extracted with acetone and the solution was mixed with 5 × SDS sample buffer (to final concentration of 2 × SDS sample buffer). The proteins in LDs and solution were then analyzed by Western blotting with the indicated antibodies. (C) ER and mitochondrial proteins in LD subpopulations. The total LD fraction was isolated from CHO K2 cells, mixed with 2.5 M sucrose to final concentration of 1.25 M, overlaid with a discontinuous sucrose density gradient, and the gradient was centrifuged at 500 g for 20 min. Fractions from the top of the gradient, 1 ml each, were collected, and the proteins from those fractions were precipitated using trichloroacetic acid (final concentration of 7.2%). The precipitated proteins were separated by SDS-PAGE, and stained with Colloidal Blue or blotted with the indicated antibodies.

Total LDs and cytosol were isolated from CHO K2 cells. The total LDs were further fractionated by size using differential centrifugation (Materials and Methods). LD fractions 1, 2 and 3 were collected. (A) LD size of the subpopulations. LD fractions were processed for fixation, dehydration and infiltration, and finally embedded in Embed 812. Then the ultra-thin sections of the three fractions were prepared and analyzed by EM. Bar = 1 μm. (B) ER and mitochondrial markers in the subpopulations. Proteins of these fractions were extracted with chloroform:acetone (300 μl:700 μl), separated using SDS-PAGE, analyzed by Western blotting (equal amount of protein per lane) with the indicated antibodies, and stained by Colloidal Blue. (C) Protein recruitment to the subpopulation. These three fractions were incubated with Buffer B (Con) or cytosol plus 1 mM GTPγs (C + G) for 1 h at 37 °C. LDs were then re-isolated and the proteins were extracted with acetone and analyzed by Western blotting with the indicated antibodies. (D) Protein composition of the subpopulations. The proteins of the fraction 1 and 3 were extracted with chloroform:acetone (300 μl:700 μl) and analyzed by silver staining and Western blotting (equal amount of protein per lane) with the indicated antibodies.

LDs from Huh7 cells were fractionated by size using differential centrifugation (Materials and Methods). Four LD fractions were collected. (A) EM to determine LD size. Huh7 cells and the four LD fractions were fixed, dehydrated and infiltrated and finally embedded for ultra-thin sectioning. The 70 nm ultra-thin sections were prepared, and the ultra-structures were analyzed by EM. (B) Protein composition of the subpopulations. Proteins of the four LD fractions were extracted with chloroform:acetone (300 μl:700 μl) and analyzed by silver staining and Western blotting (equal amount of protein per lane) with the indicated antibodies.

LDs from mouse liver were fractionated by size using differential centrifugation (Materials and Methods). Four LD fractions were collected. (A) LD size of the subpopulations. LD fractions were viewed under light microscope (upper panel). The LDs were incubated with 1% Triton for 30 min to reduce aggregation. After removing Triton, the LDs were viewed and the images were acquired at 400-fold magnification. The LD size was measured by a Delsa Nano C particle analyzer. (B) Protein profile of the subpopulations. The proteins of LD subpopulations were extracted and analyzed by silver staining. (C) ER and mitochondrial markers in the subpopulations. Proteins from the subpopulations were analyzed by Western blotting with indicated antibodies (equal amount of protein per lane). (D,E) Neutral lipid analysis. Lipids of the subpopulations from mouse liver (panel D) and CHO K2 cells (panel E) were separated by TLC with a solvent of n-hexane-diethyl ether-acetic acid (80:20:1, v/v/v). TLC plate was then stained in iodine vapor after being dried at room temperature.

LDs from mouse BAT were fractionated by size using differential centrifugation (Materials and Methods). Four LD fractions were collected. (A) EM to determine LD size. The four LD fractions were processed for positive staining and the sizes of the LDs were analyzed by EM. Bar = 2 μm. (B) Protein profile of the subpopulations. Proteins of LD subpopulations were extracted with chloroform:acetone (300 μl:700 μl) and analyzed by Western blotting with the indicated antibodies and silver staining (equal amount of protein per lane).

LDs and cytosol were isolated from CHO K2 cells, and the LDs were further separated into three subpopulations using differential centrifugation (Materials and Methods). Fraction 3, the LD subpopulation nearly depleted of ER, was used for an in vitro fatty acid incorporation assay. The faction 3 LDs had first been incubated with [³H]OA and unlabeled OA at 4 °C for 6 h. (A) Requirement of ATP and cytosol for fatty acid incorporation. The OA-associated LDs were then aliquoted and incubated with 2 mM ATP alone or 100 μl cytosol in the presence or absence of 2 mM ATP at 37 °C for 2 h. (B) Requirement of ATP and CoA for fatty acid incorporation. The OA-associated LDs were then aliquoted and incubated with 2 mM CoA alone or the indicated concentrations of CoA (mM) in the presence or absence of 2 mM ATP at 37 °C for 2 h. (C–F) Time-dependent fatty acid incorporation. The OA-associated LDs were then aliquoted and incubated with the indicated concentration of ATP (μM) at 37 °C for 2 h in the presence of 100 μl cytosol (C), or with 2 mM ATP in the presence or absence of 2 mM CoA at 37 °C for the indicated times (D–F). After incubation, LDs were re-isolated, washed 3 times with Buffer B, and lipids extracted with acetone:chloroform (2:1, v/v) and separated using TLC. The bands of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and triacylglycerol (TAG) were scraped from TLC plate and counted with scintillation fluid.