Isolation of LDs from zebrafish liver. A Flowchart of isolation of LDs from zebrafish liver. Livers of adult zebrafishes were collected and homogenized, and the fraction of PNS was collected and subjected to centrifuge at 500 g, 2,000 g and 20,000 g. After centrifugation, the white layer on the top of the gradient was collected as LD fraction, the pellet at the bottom was TM, and the middle solution was collected as Cyto. The LD fraction was then washed three times and kept in an Eppendorf tube for further usage. B Isolated LDs were first analyzed by confocal microscopy with LipidTOX Red staining, DIC imaging, and merged images. Bar = 10 μm. C The size of isolated LDs was measured by a Delsa Nano C particle analyzer

Proteomic analysis of isolated LDs from zebrafish liver. A Proteins were separated by SDS-PAGE after extracting from LD, TM, Cyto, and PNS fractions, and then stained by colloidal blue. Red arrowheads marked unique protein bands of LDs. B Proteins of LDs were precipitated by acetone and then subjected to nano-LC-ESI-LTQ Orbitrap XL MS/MS analysis. The Venn diagram showed the overlap of two independent mass spectrum results. C The 259 proteins of LDs identified in two samples were categorized into 11 groups: perilipin family, lipid metabolism, mitochondria, endoplasmic reticulum, cytoskeleton, chaperone, cell signal, membrane trafficking, protein translation and modification degradation, other protein and unknown function protein. D Proteins of LDs of two independent mass spectrum results were categorized into previously reported proteins and newly identified proteins in this study

Localization of Plin2 and Plin3 of zebrafish in Huh7 cells. A The amino acid sequence alignment of Plin2 of human and zebrafish. The amino acid sequences were compared by Clustal X and visualized by ESPript3.0. B The amino acid sequence alignment of Plin3 of human and zebrafish. The amino acid sequences were compared by Clustal X and visualized by ESPript3.0. The black frame showed the absence of sequence in zebrafish Plin3. C Zebrafish plin2 was cloned into GFP plasmid and transiently overexpressed in Huh7 cells. The cells were imaged by an Olympus FV1200 confocal microscope. Upper panels: the vector GFP overexpressed in Huh7 cells; Middle panels: Plin2-GFP overexpressed in Huh7 cells; Lower panels: Plin2-GFP overexpressed in Huh7 cells and treated with 100 μmol/L OA for 6 h. Green: GFP; Red: LipidTOX Red; Blue: Hoechst. Bar = 10 μm. D Zebrafish plin3 was cloned into GFP plasmid and transiently overexpressed in Huh7 cells. The cells were imaged by an Olympus FV1200 confocal microscope. Upper panels: the vector GFP overexpressed in Huh7 cells; Middle panels: Plin3-GFP overexpressed in Huh7 cells; Lower panels: Plin3-GFP overexpressed in Huh7 cells and treated with 100 μmol/L OA for 6 h. Green: GFP; Red: LipidTOX Red; Blue: Hoechst. Bar = 10 μm. E Helical wheel projection of sequences of human PLIN3 in the black frame was predicted by HeliQuest

Isolation of LDs from Carassius auratus liver. A Livers of Carassius auratus were collected and homogenized by dounce, the fraction of PNS was collected after centrifugation at 500 g and subjected to separation of LD fraction. After centrifugation with the indicated speed, the white layer on the top of the gradient was collected as LD fraction, the pellet at the bottom was resuspended as TM, and the middle solution was collected as Cyto. The LD fraction was then washed three times and kept in an Eppendorf tube as isolated LDs for further usage. B Isolated LDs were first analyzed by confocal microscopy with LipidTOX Red staining, DIC imaging, and merged images. Bar = 10 μm. C The size of isolated LDs was measured by a Delsa Nano C particle analyzer

Proteomic analyses of isolated LDs from Carassius auratus liver. A Proteins were separated by SDS-PAGE after extracting from LD, TM, Cyto and PNS fractions, and stained by silver staining. B Proteins of LDs were precipitated by acetone and then subjected to nano-LC-ESI-LTQ Orbitrap XL MS/MS analysis. The Venn diagram showed the overlap of two independent mass spectrum results. C Proteins of LDs of two independent mass spectrum results were categorized into previously reported proteins and newly identified proteins in this study. D The 267 proteins of LDs identified in two samples were categorized into 12 groups: perilipin family, lipid metabolism, mitochondria, endoplasmic reticulum, cytoskeleton, chaperone, cell signal, membrane trafficking, protein translation and modification degradation, ribosome, other protein and unknown function protein